12 human secreted proteins

ABSTRACT

The present invention relates to 12 novel human secreted proteins and isolated nucleic acids containing the coding regions of the genes encoding such proteins. Also provided are vectors, host cells, antibodies, and recombinant methods for producing human secreted proteins. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to these novel human secreted proteins.

This application is a division of U.S. application Ser. No. 09/984,130,filed Oct. 29, 2001 (now abandoned), which claims benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 60/243,792, filed onOct. 30, 2000, and which is also a continuation-in-part of U.S.application Ser. No. 09/836,353, filed Apr. 18, 2001 (now abandoned),which claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication No. 60/198,407, filed Apr. 19, 2000, and which is acontinuation-in-part of PCT International Application No. PCT/US99/25031(published in English), filed Oct. 27, 1999, which claims benefit under35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/105,971, filedOct. 28, 1998. Each of the above referenced patent applications ishereby incorporated by reference herein.

STATEMENT UNDER 37 C.F.R. § 1.77(b)(4)

This application refers to a “Sequence Listing” listed below, which isprovided as an electronic document on two identical compact discs(CD-R), labeled “Copy 1” and “Copy 2.” These compact discs each containthe file “PF489P2D1 Seq list.txt” (288,736 bytes, created on Oct. 5,2005), which is hereby incorporated by reference in its entirety herein.The Sequence Listing may be viewed on an IBM-PC machine running theMS-Windows operating system.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides and thepolypeptides encoded by these polynucleotides, uses of suchpolynucleotides and polypeptides, and their production.

BACKGROUND OF THE INVENTION

Unlike bacterium, which exist as a single compartment surrounded by amembrane, human cells and other eucaryotes are subdivided by membranesinto many functionally distinct compartments. Each membrane-boundedcompartment, or organelle, contains different proteins essential for thefunction of the organelle. The cell uses “sorting signals,” which areamino acid motifs located within the protein, to target proteins toparticular cellular organelles.

One type of sorting signal, called a signal sequence, a signal peptide,or a leader sequence, directs a class of proteins to an organelle calledthe endoplasmic reticulum (ER). The ER separates the membrane-boundedproteins from all other types of proteins. Once localized to the ER,both groups of proteins can be further directed to another organellecalled the Golgi apparatus. Here, the Golgi distributes the proteins tovesicles, including secretory vesicles, the cell membrane, lysosomes,and the other organelles.

Proteins targeted to the ER by a signal sequence can be released intothe extracellular space as a secreted protein. For example, vesiclescontaining secreted proteins can fuse with the cell membrane and releasetheir contents into the extracellular space—a process called exocytosis.Exocytosis can occur constitutively or after receipt of a triggeringsignal. In the latter case, the proteins are stored in secretoryvesicles (or secretory granules) until exocytosis is triggered.Similarly, proteins residing on the cell membrane can also be secretedinto the extracellular space by proteolytic cleavage of a “linker”holding the protein to the membrane.

Despite the great progress made in recent years, only a small number ofgenes encoding human secreted proteins have been identified. Thesesecreted proteins include the commercially valuable human insulin,interferon, Factor VIII, human growth hormone, tissue plasminogenactivator, and erythropoeitin. Thus, in light of the pervasive role ofsecreted proteins in human physiology, a need exists for identifying andcharacterizing novel human secreted proteins and the genes that encodethem. This knowledge will allow one to detect, to treat, and to preventmedical disorders by using secreted proteins or the genes that encodethem.

SUMMARY OF THE INVENTION

The present invention relates to novel polynucleotides and the encodedpolypeptides. Moreover, the present invention relates to vectors, hostcells, antibodies, and recombinant and synthetic methods for producingthe polypeptides and polynucleotides. Also provided are diagnosticmethods for detecting disorders and conditions related to thepolypeptides and polynucleotides, and therapeutic methods for treatingsuch disorders and conditions. The invention further relates toscreening methods for identifying binding partners of the polypeptides.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-C show the nucleotide (SEQ ID NO:11) and deduced amino acidsequence (SEQ ID NO:29) of this protein.

FIG. 2 shows the regions of similarity between the amino acid sequencesof SEQ ID NO:29, the Xenopus laevis tail resorption protein (gi|1234787)(SEQ ID NO:48), and the Hedgehog Interacting Protein (“HIP”;gi|AAD31172.1) (SEQ ID NO:49).

FIG. 3 shows an analysis of the amino acid sequence of SEQ ID NO: 29.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

FIGS. 4A-C shows the nucleotide (SEQ ID NO:12) and deduced amino acidsequence (SEQ ID NO:30) of TIDE. Predicted amino acids from about 1 toabout 23 constitute the predicted signal peptide (amino acid residuesfrom about 1 to about 23 in SEQ ID NO:30) and are represented by theunderlined amino acid regions; amino acids from about 108 to about 136,from about 195 to about 223, from about 291 to about 319, from about 379to about 407, and/or from about 465 to about 493 constitute thepredicted EGF-like domain signature 1 and 2 domains (amino acids fromabout 108 to about 136, from about 195 to about 223, from about 291 toabout 319, from about 379 to about 407, and/or from about 465 to about493 in SEQ ID NO:30) and are represented by the double underlined aminoacids; and amino acids from about 55 to about 89, from about 97 to about129, from about 142 to about 175, from about 186 to about 216, fromabout 228 to about 261, from about 281 to about 314, from about 327 toabout 359, from about 368 to about 398, from about 417 to about 448,and/or from about 455 to about 487 constitute the predicted integrinsbeta chain cysteine-rich domains (amino acids from about 55 to about 89,from about 97 to about 129, from about 142 to about 175, from about 186to about 216, from about 228 to about 261, from about 281 to about 314,from about 327 to about 359, from about 368 to about 398, from about 417to about 448, and/or from about 455 to about 487 in SEQ ID NO:30) andare represented by the shaded amino acids.

FIG. 5 shows the regions of similarity between the amino acid sequencesof the Ten Integrin Domains with EGF homology (TIDE) protein (SEQ IDNO:30) and the human integrin beta-8 subunit (SEQ ID NO: 67).

FIG. 6 shows an analysis of the Ten Integrin Domains with EGF homology(TIDE) amino acid sequence. Alpha, beta, turn and coil regions;hydrophilicity and hydrophobicity; amphipathic regions; flexibleregions; antigenic index and surface probability are shown.

FIGS. 7A-B show the nucleotide (SEQ ID NO:13) and deduced amino acidsequence (SEQ ID NO:31) of the Intestine derived extracellular protein.Predicted amino acids from about 1 to about 27 constitute the predictedsignal peptide (amino acid residues from about 1 to about 27 in SEQ IDNO:31) and are represented by the underlined amino acid regions; andamino acids from about 122 to about 138 constitute the predictedtransmembrane domain (amino acid residues from about 122 to about 138 inSEQ ID NO:31) and are represented by the double-underlined amino acids.

FIG. 8 shows the regions of similarity between the amino acid sequencesof the Intestine derived extracellular protein SEQ ID NO:31, and theRAMP3 protein (gi|4587099) (SEQ ID NO: 75).

FIG. 9 shows an analysis of the amino acid sequence of SEQ ID NO: 31.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

FIGS. 10A-B shows the nucleotide (SEQ ID NO:14) and deduced amino acidsequence (SEQ ID NO:32) of the retinal specific protein. Predicted aminoacids from about 1 to about 21 constitute the predicted signal peptide(amino acid residues from about 1 to about 21 in SEQ ID NO:32) and arerepresented by the underlined amino acid regions.

FIG. 11 shows the regions of similarity between the amino acid sequencesof the retinal specific protein SEQ ID NO:32, and the Gallus gallusproteoglycan (SEQ ID NO:79).

FIG. 12 shows an analysis of the amino acid sequence of SEQ ID NO: 32.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

FIGS. 13A-C shows the nucleotide (SEQ ID NO:15) and deduced amino acidsequence (SEQ ID NO:33) of the CD33-like protein. Predicted amino acidsfrom about 1 to about 16 constitute the predicted signal peptide (aminoacid residues from about 1 to about 16 in SEQ ID NO:33) and arerepresented by the underlined amino acid regions; and amino acids fromabout 496 to about 512 constitute the predicted transmembrane domain(amino acid residues from about 496 to about 512 in SEQ ID NO:33) andare represented by the double-underlined amino acid regions.

FIG. 14 shows the regions of similarity between the amino acid sequencesof the CD33-like protein SEQ ID NO:33, and the CD33L1 protein (gi|88178)(SEQ ID NO: 92).

FIG. 15 shows an analysis of the amino acid sequence of SEQ ID NO:33.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

FIGS. 16A-B show the nucleotide (SEQ ID NO:16) and deduced amino acidsequence (SEQ ID NO:34) of CD33-like 3. Predicted amino acids from about1 to about 18 constitute the predicted signal peptide (amino acidresidues from about 1 to about 18 in SEQ ID NO:34) and are representedby the underlined amino acid regions; and amino acids from about 360 toabout 376 constitute the predicted transmembrane domain (amino acidsfrom about 360 to about 376 in SEQ ID NO:34) and are represented by thedouble underlined amino acids.

FIG. 17 shows the regions of similarity between the amino acid sequencesof the CD33-like 3 protein (SEQ ID NO:34) and the human CD33L1 protein(SEQ ID NO:99).

FIG. 18 shows an analysis of the CD33-like 3 amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

FIGS. 19A-F show the nucleotide (SEQ ID NO:17) and deduced amino acidsequence (SEQ ID NO:35) of a11. Predicted amino acids from about 1 toabout 22 constitute the predicted signal peptide (amino acid residuesfrom about 1 to about 22 in SEQ ID NO:35) and are represented by theunderlined amino acid regions; amino acids from about 666 to about 682,and/or amino acids from about 1145 to about 1161 constitute thepredicted transmembrane domains (amino acids from about 666 to about682, and/or amino acids from about 1145 to about 1161 in SEQ ID NO:35)and are represented by the double underlined amino acids; and aminoacids from about 64 to about 96 constitute the predicted immunoglobulinand major histocompatibility complex protein domain (amino acids fromabout 64 to about 96 in SEQ ID NO:35) and are represented by the boldamino acids.

FIG. 20 shows the regions of similarity between the amino acid sequencesof the integrin alpha 11 subunit (a11) protein (SEQ ID NO:35) and thehuman integrin alpha 1 subunit (SEQ ID NO: 103).

FIG. 21 shows an analysis of the integrin alpha 11 subunit (a11) aminoacid sequence. Alpha, beta, turn and coil regions; hydrophilicity andhydrophobicity; amphipathic regions; flexible regions; antigenic indexand surface probability are shown.

FIGS. 22A-C show the nucleotide (SEQ ID NO:19) and deduced amino acidsequence (SEQ ID NO:37) of BBIR II. Predicted amino acids from about 1to about 17 constitute the predicted signal peptide (amino acid residuesfrom about 1 to about 17 in SEQ ID NO:37) and are represented by theunderlined amino acid regions.

FIG. 23 shows the regions of similarity between the amino acid sequencesof the Butyrophlin and B7-like IgG superfamily receptor (BBIR II)protein (SEQ ID NO:37) and the bovine butyrophilin precursor (SEQ IDNO:121)

FIG. 24 shows an analysis of the integrin alpha 11 subunit (BBIR II)amino acid sequence.

FIGS. 25 A-B show the nucleotide (SEQ ID NO:20) and deduced amino acidsequence (SEQ ID NO:38) of the present invention. Predicted amino acidsfrom about 1 to about 19 constitute the predicted signal peptide (aminoacid residues from about 1 to about 19 in SEQ ID NO:38) and arerepresented by the underlined amino acid regions; amino acids from about162 to about 188 constitutes the predicted serine protease histidineactive site domain (amino acids residues from about 162 to about 188 inSEQ ID NO:38) and are represented by the double underlined amino acidregions; and amino acid residue 175 (amino acid residue 175 in SEQ IDNO:38) constitutes the predicted histidine active site residue and isrepresented by the bold amino acid.

FIG. 26 shows the regions of similarity between the amino acid sequencesof the present invention SEQ ID NO:38, and the Human Pancreatic Elastase2 protein (gi|219620)(SEQ ID NO: 127).

FIG. 27 shows an analysis of the amino acid sequence of SEQ ID NO:38.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

FIGS. 28A-B shows the nucleotide (SEQ ID NO:21) and deduced amino acidsequence (SEQ ID NO:39) of the present invention. Predicted amino acidsfrom about 1 to about 44 constitute the predicted signal peptide (aminoacid residues from about 1 to about 44 in SEQ ID NO:39) and arerepresented by the underlined amino acid regions.

FIG. 29 shows the regions of similarity between the amino acid sequencesof the present invention SEQ ID NO:39, the human poliovirus receptorprotein (gi|1524088) (SEQ ID NO: 138), the human class-I MHC-restrictedT cell associated molecule (WO9634102) (SEQ ID NO:144), and the Gallusgallus thymocyte activation and developmental protein (gb|AAB88491.1)(SEQ ID NO: 145).

FIG. 30 shows an analysis of the amino acid sequence of SEQ ID NO:39.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

FIG. 31 shows the nucleotide (SEQ ID NO:22) and deduced amino acidsequence (SEQ ID NO:40) of the present invention. Predicted amino acidsfrom about 1 to about 23 constitute the predicted signal peptide (aminoacid residues from about 1 to about 23 in SEQ ID NO:40) and arerepresented by the underlined amino acid regions.

FIG. 32 shows the regions of similarity between the amino acid sequencesof the present invention SEQ ID NO:40 and the human FAP protein(gi|1890647) (SEQ ID NO:146).

FIG. 33 shows an analysis of the amino acid sequence of SEQ ID NO:40.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

FIG. 34 shows the nucleotide (SEQ ID NO:18) and deduced amino acidsequence (SEQ ID NO:36) of htag7. Predicted amino acids from about 1 toabout 21 constitute the predicted signal peptide (amino acid residuesfrom about 1 to about 21 in SEQ ID NO:36) and are represented by theunderlined amino acid regions; and amino acids from about 34 to about117 constitute the predicted PGRP-like domain (amino acids from about 34to about 117 in SEQ ID NO:36) and are represented by the doubleunderlined amino acids.

FIG. 35 shows the regions of similarity between the amino acid sequencesof the htag7 protein (SEQ ID NO:36) and the mouse tag7 protein (SEQ IDNO:114).

FIG. 36 shows an analysis of the htag7 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown.

FIG. 37 shows the predicted protein sequence of CD33-likeSV protein(identified in FIG. 37 as “Siglec-10”) and alignment withclosely-related Siglecs. Alignment was performed with the ClustalWmultiple sequence alignment program. Residues that are identical in morethan half the proteins are boxed in black, similar residues in grey.Asterisks indicate positions of the cysteine residues characteristic ofSiglecs. Filled circles overlay residues important for sialic acidbinding. Vertical lines indicate positions of intron-exon boundaries, asdeduced from the sequence of the gene encoding CD33-likeSV. Positions ofthe domain boundaries, transmembrane region, cytoplasmic tail (encodedby two exons) and the tyrosine based motifs are indicated. Genbankaccession numbers are as follows: CD33/Siglec-3, Siglec-5: AAD50978;Siglec-6, NP001236; Siglec-7 AAF12759; Siglec-8, AAF27622; and Siglec-9,AAF87223.

FIG. 38 provides a phylogenetic analysis of CD33-related Siglecs andCD33-likeSV protein (identified in FIG. 38 as “Siglec-10”). The leaderpeptide, domain 1, and domain 2, or the transmembrane and cytoplasmictails were aligned using the Clustal W multiple sequence alignmentprogram and analyzed for phylogenetic relationship using the PHYLIP 3.6.Unrooted phylograms were constructed using the neighbor joining method.

FIG. 39 provides a sequential deletion model for evolution ofCD33-related Siglecs. Based on the phylogenetic analysis and sequencecomparisons, this model predicts that MAG and CD33-likeSV (identified inFIG. 39 as “Siglec-10”) are both derived from a common 4-domainprogenitor. CD33-likeSV then gave rise to Siglec-5 involving a deletionof the exons encoding domain 3 and its associated linker. Siglec-5 thengave rise to a three domain siglec by deletion of domain 4. The other3-domain Siglecs then arose through gene duplication. CD33 may have beenderived in a single deletion event either from Siglec-5 or one of thethree domain Siglecs.

FIGS. 40A & B provides the localization and expression of theCD33-likeSV gene.

Human lymphocyte metaphase spreads were hybridized with a 3 kbbiotinylated insert from HEONM10 followed by fluorescein-avidin and thechromosomes counterstained with propidium iodide. The digital image isreversed to illustrate the hybridization signals (arrows) on the longarm of chromosome 19. The position of CD33-likeSV on chromosome 19 bandq13.3 is also shown schematically.

Northern blot analysis of CD33-likeSV mRNA in human tissues. Each laneof the Multiple Tissue Northern (MTN) Blot (Clontech) containsapproximately 2 μg poly A+ RNA from the tissue indicated and isnormalized for levels of β-actin mRNA. A major form of CD33-likeSV mRNAis seen at around 3.0 kb in most tissues.

FIG. 41 disclosed the binding of CD33-likeSV (identified in FIG. 41 as“Siglec-10”) expressed on COS cells to polyacrylamide conjugates. CR1was included as a negative control to measure non-specific binding.Three days after transient transfection, COS cells expressing theindicated proteins were incubated with biotinylated polyacrylamide (PAA)glycoconjugates linked either to 3′ sialyllactose (2,3-PAA) or 6′sialyllactose (2,6-PAA) or lactose (Lac-PAA) at 20 μg/ml or with bufferalone. Unbound conjugate was washed off and binding detected with¹²⁵I-streptavidin. Data show means standard deviations of quadruplicatesand are representative of 3 experiments performed.

FIGS. 42A, B & C show the expression of CD33-likeSV (identified in FIG.42 as “Siglec-10”) on human peripheral blood leukocyte subsets.

FACS histograms showing expression of CD33-likeSV on granulocytes,monocytes and lymphocytes, gated in each case according to theircharacteristic side and forward scatter properties. Thick lines showstaining with affinity purified mouse anti-CD33-likeSV polyclonalantibody. Thin lines show staining in the presence of mouse IgG used asa negative control. CD33-likeSV is expressed on a minor subset ofgranulocytes, most monocytes and a subset of lymphocytes

Double labeling of granulocytes with anti-CD 16 (neutrophils) andanti-CD33-likeSV mAb compared with anti-Siglec-8 mAb. Compared to theisotype matched control mAb, CD33-likeSV shows clear labeling of theeosinophils and some of the neutrophils are also weakly stained. Valuesrepresent the percentages of total granulocytes analyzed.

Double labeling of the lymphocyte fraction with antibodies to CD 19 (Bcells), CD3 (pan T cell), CD4 and CD8 (T cell subsets) and CD56 (NKcells). CD33-likeSV is expressed by most CD 19+ B cells and a smallsubset of CD16+ cells that do not express the CD56 natural killer cellmarker. Values represent the percentages of the total lymphocytesanalyzed. Similar results were obtained using the mouse anti-CD33-likeSVmAb, 5G6.

FIG. 43 provides a molecular characterization of CD33-likeSV (identifiedin FIG. 43 as “S10”). Stably-transfected CHO cells expressingCD33-likeSV, wild-type CHO cells, or Daudi cells were surfacebiotinylated, lysed and immunoprecipitations performed with mouseanti-CD33-likeSV polyclonal antibody. Precipitates were run either underreducing or non-reducing conditions on 4-12% gradient SDS polyacrylamidegels, transferred to nitrocellulose and probed with streptavidin-horseradish peroxidase. CD33-likeSV migrates as a single monomeric species ataround 120 kDa in CHO cells and around 100 kDa in Daudi cells.

FIG. 44 shows an analysis of the amino acid sequence of CD33-likeSVprotein (SEQ ID NO: 149). Alpha, beta, turn and coil regions;hydrophilicity and hydrophobicity; amphipathic regions; flexibleregions; antigenic index and surface probability are shown.

DETAILED DESCRIPTION

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.The term “isolated” does not refer to genomic or cDNA libraries, wholecell total or mRNA preparations, genomic DNA preparations (includingthose separated by electrophoresis and transferred onto blots), shearedwhole cell genomic DNA preparations or other compositions where the artdemonstrates no distinguishing features of the polynucleotide/sequencesof the present invention.

In the present invention, a “secreted” protein refers to those proteinscapable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as thoseproteins released into the extracellular space without necessarilycontaining a signal sequence. If the secreted protein is released intothe extracellular space, the secreted protein can undergo extracellularprocessing to produce a “mature” protein. Release into the extracellularspace can occur by many mechanisms, including exocytosis and proteolyticcleavage.

In specific embodiments, the polynucleotides of the invention are atleast 15, at least 30, at least 50, at least 100, at least 125, at least500, or at least 1000 continuous nucleotides but are less than or equalto 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5 kb, 5 kb, 2.5 kb,2.0 kb, or 1 kb, in length. In a further embodiment, polynucleotides ofthe invention comprise a portion of the coding sequences, as disclosedherein, but do not comprise all or a portion of any intron. In anotherembodiment, the polynucleotides comprising coding sequences do notcontain coding sequences of a genomic flanking gene (i.e., 5′ or 3′ tothe gene of interest in the genome). In other embodiments, thepolynucleotides of the invention do not contain the coding sequence ofmore than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1genomic flanking gene(s).

As used herein, a “polynucleotide” refers to a molecule having a nucleicacid sequence contained in SEQ ID NO:X or the cDNA contained within theclone deposited with the ATCC. For example, the polynucleotide cancontain the nucleotide sequence of the full length cDNA sequence,including the 5′ and 3′ untranslated sequences, the coding region, withor without the signal sequence, the secreted protein coding region, aswell as fragments, epitopes, domains, and variants of the nucleic acidsequence. Moreover, as used herein, a “polypeptide” refers to a moleculehaving the translated amino acid sequence generated from thepolynucleotide as broadly defined.

In the present invention, the full length sequence identified as SEQ IDNO:X was often generated by overlapping sequences contained in multipleclones (contig analysis). A representative clone containing all or mostof the sequence for SEQ ID NO:X was deposited with the American TypeCulture Collection (“ATCC”). As shown in Table XIV, each clone isidentified by a cDNA Clone ID (Identifier) and the ATCC Deposit Number.The ATCC is located at 10801 University Boulevard, Manassas, Va.20110-2209, USA. The ATCC deposit was made pursuant to the terms of theBudapest Treaty on the international recognition of the deposit ofmicroorganisms for purposes of patent procedure.

A “polynucleotide” of the present invention also includes thosepolynucleotides capable of hybridizing, under stringent hybridizationconditions, to sequences contained in SEQ ID NO:X the complementthereof, or the cDNA within the clone deposited with the ATCC.“Stringent hybridization conditions” refers to an overnight incubationat 42 degree C. in a solution comprising 50% formamide, 5×SSC (750 mMNaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65 degree C.

Also contemplated are nucleic acid molecules that hybridize to thepolynucleotides of the present invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37 degree C. ina solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blocking DNA;followed by washes at 50 degree C. with 1×SSPE, 0.1% SDS. In addition,to achieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a

complementary stretch of T (or U) residues, would not be included in thedefinition of “polynucleotide,” since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-stranded cDNA clonegenerated using oligo dT as a primer).

The polynucleotide of the present invention can be composed of anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. For example, polynucleotides can becomposed of single- and double-stranded DNA, DNA that is a mixture ofsingle- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions. In addition, the polynucleotide can be composed oftriple-stranded regions comprising RNA or DNA or both RNA and DNA. Apolynucleotide may also contain one or more modified bases or DNA or RNAbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

The polypeptide of the present invention can be composed of amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and may contain amino acids other than the 20gene-encoded amino acids. The polypeptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are well described in basic texts and in more detailedmonographs, as well as in a voluminous research literature.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.Polypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic polypeptides may result fromposttranslation natural processes or may be made by synthetic methods.Modifications include acetylation, acylation, ADP-ribosylation,amidation, covalent attachment of flavin, covalent attachment of a hememoiety, covalent attachment of a nucleotide or nucleotide derivative,covalent attachment of a lipid or lipid derivative, covalent attachmentof phosphotidylinositol, cross-linking, cyclization, disulfide bondformation, demethylation, formation of covalent cross-links, formationof cysteine, formation of pyroglutamate, formylation,gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation,iodination, methylation, myristoylation, oxidation, pegylation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. (See, forinstance, PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993); POSTTRANSLATIONALCOVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press,New York, pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646(1990); Rattan et al., Ann NY Acad Sci 663:48-62 (1992).)

“SEQ ID NO:X” refers to a polynucleotide sequence while “SEQ ID NO:Y”refers to a polypeptide sequence, both sequences identified by aninteger specified in Table XIV.

“A polypeptide having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a polypeptide of the present invention, including matureforms, as measured in a particular biological assay, with or withoutdose dependency. In the case where dose dependency does exist, it neednot be identical to that of the polypeptide, but rather substantiallysimilar to the dose-dependence in a given activity as compared to thepolypeptide of the present invention (i.e., the candidate polypeptidewill exhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to thepolypeptide of the present invention.)

Polynucleotides and Polypeptides of the Invention

Features of Protein Encoded by Gene No: 1

Translation products corresponding to this gene share sequence homologywith a protein from Xenopus laevis that is described as upregulated inresponse to thyroid hormone in tadpoles, and is thought to be importantin the tail resorption process during Xenopus laevis metamorphosis (SeeProc. Natl. Acad. Sci. USA (Mar. 5, 1996):93(5):1924-9, which is hereinincorporated by reference). In addition, translation productscorresponding to this gene share sequence homology with a recentlydescribed group of proteins, called hedgehog interacting proteins (HIPs)(See International Publication No. WO98/12326, which is hereinincorporated by reference). These proteins bind to hedgehog polypeptidessuch as Shh and Dhh with high affinity (Kd approx. 1 nM). HIPs exhibitspatially and temporally restricted expression domains indicative ofimportant roles in hedgehog-mediated induction. They regulatedifferentiation of neuronal cells, regulate survival of differentiatedneuronal cells, proliferation of chondrocytes, proliferation oftesticular germ line cells and/or expression of patched or hedgehoggenes. The biological activity of this polypeptide is assayed bytechniques known in the art, otherwise disclosed herein and as describedin International Publication No. WO98/12326, which is hereinincorporated by reference.

Preferred polypeptides of the invention comprise, or alternativelyconsist of, the following amino acid sequence:MLRTSTPNLCGGLHCRAPWLSSGILCLCLIFLLGQVGLLQGHPQCLDYGPPFQPPLHLEFCSDYESFGCCDQHKDRRIAARYWDIMEYFDLKRHELCGDYIKDILCQECSPYAAHLYDAENTQTPLRNLPGLCSDYCSAFHSNCHSAISLLTNDRGLQESHGRDGTRFCHLLDLPDKDYCFPNVLRNDYLNRHLGMVAQDPQGCLQLCLSEVANGLRNPVSMVHAGDGTHRFFVAEQVGVVWVYLPDGSRLEQPFLDLKNIVLTTPWIGDERGFLGLAFHPKFRHNRKFYIYYSCLDKKKVEKIRISEMKVSRADPNKADLKSERVILEIEEPASNHNGGQLLFGLDGYMYIFTGDGGQAGDPFGLFGNAQNKSSLLGKVLRIDVNRAGSHGKRYRVPSDNPFVSEPGAHPAIYAYGIRNMWRCAVDRGDPITRQGRGRIFCGDVGQNRFEEVDLILKGGNYGWRAKEGFACYDKKLCHNASLDDVLPIYAYGHAVGKSVTGGYVYRGCESPNLNGLYIFGDFMSGRLMALQEDRKNKKWKKQDLCLGSTTSCAFPGLISTHSKFIISFAEDEAGELYFLATSYPSAYAPRGSIYKFVDPSRRAPPGKCKYKPVPVRTKSKRIPFRPLAKTVLDLLKEQSEKAARKSSSATLASGPAQGLSEKGSSKKLASPTSSKNTLRGPGTKKKARVGPHVRQGKRRKSLKSHSGRMRPSAEQKRAGRSLP (SEQ ID NO: 47). Also preferred are polypeptidescomprising the mature polypeptide which is predicted to consist ofresidues 42-724 of the foregoing sequence, and biologically activefragments of the mature polypeptide. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g. fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,or all sixteen of the immunogenic epitopes shown in SEQ ID NO: 29 asresidues: Asp-52 to Glu-57, Arg-89 to Tyr-95, Asp-102 to Glu-107,Ser-117 to Ser-128, Glu-137 to Gly-145, Arg-192 to Arg-199, Val-231 toGly-243, Val-250 to Glu-256, Arg-312 to Asn-318, Glu-338 to Asp-349,Pro-405 to Lys-417, Thr-423 to Ile-428, Lys-442 to Ser-453, Glu-467 toAla-475, Thr-478 to Arg-494, and/or Pro-497 to Arg-526. Polynucleotidesencoding these polypeptides are also encompassed by the invention, asare antibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g. fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

FIGS. 1A-C show the nucleotide (SEQ ID NO:11) and deduced amino acidsequence (SEQ ID NO:29) of this protein.

FIG. 2 shows the regions of similarity between the amino acid sequencesof SEQ ID NO:29, the Xenopus laevis tail resorption protein (gi|1234787)(SEQ ID NO:48), and the Hedgehog Interacting Protein (“HIP”;gi|AAD31172.1) (SEQ ID NO:49).

FIG. 3 shows an analysis of the amino acid sequence of SEQ ID NO: 29.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the polypeptide having the aminoacid sequence shown in FIGS. 1A-C (SEQ ID NO:29), which was determinedby sequencing a cloned cDNA. The nucleotide sequence shown in FIGS. 1A-C(SEQ ID NO:11) was obtained by sequencing a cloned cDNA, which wasdeposited on Nov. 17, 1998 at the American Type Culture Collection, andgiven Accession Number 203484. The deposited gene is inserted in thepSport plasmid (Life Technologies, Rockville, Md.) using the SalI/NotIrestriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO:11 is intended DNA fragments atleast about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:11. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:11. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Representative examples of polynucleotide fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, and from about 501to about 550, and from about 551 to about 570 of SEQ ID NO:11, or thecomplementary strand thereto, or the cDNA contained in the depositedgene. In this context “about” includes the particularly recited ranges,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. In additional embodiments, thepolynucleotides of the invention encode functional attributes of thecorresponding protein.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions. The data representing the structural or functionalattributes of the protein set forth in FIG. 3 and/or Table I, asdescribed above, was generated using the various modules and algorithmsof the DNA*STAR set on default parameters. In a preferred embodiment,the data presented in columns VIII, IX, XIII, and XIV of Table I can beused to determine regions of the protein which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX, XIII, and/or XIV bychoosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

Certain preferred regions in these regards are set out in FIG. 3, butmay, as shown in Table I, be represented or identified by using tabularrepresentations of the data presented in FIG. 3. The DNA*STAR computeralgorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table I). The tabular format of the data in FIG. 3 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 3 and in Table Iinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS. 1A-C(SEQ ID NO:29). As set out in FIG. 3 and in Table I, such preferredregions include Garnier-Robson alpha-regions, beta-regions,turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions,and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woodshydrophobic regions, Eisenberg alpha- and beta-amphipathic regions,Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenicindex and Emini surface-forming regions. Even if deletion of one or moreamino acids from the N-terminus of a protein results in modification ofloss of one or more biological functions of the protein other functionalactivities (e.g., biological activities, ability to multimerize, etc.)may still be retained. For example, the ability of shortened muteins toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptides generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the N-terminus. Whether a particular polypeptide lackingN-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a mutein with alarge number of deleted N-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence shown in FIGS. 1A-C, up to the alanine residue at positionnumber 524 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-524 of FIGS. 1A-C, where n1 is aninteger from 1 to 524 corresponding to the position of the amino acidresidue in FIGS. 1A-C (which is identical to the sequence shown as SEQID NO:29). N-terminal deletions of the polypeptide of the inventionshown as SEQ ID NO:29 include polypeptides comprising the amino acidsequence of residues: V-2 to P-529; A-3 to P-529; Q-4 to P-529; D-5 toP-529; P-6 to P-529; Q-7 to P-529; C-9 to P-529; L-10 to P-529; Q-11 toP-529; L-12 to P-529; C-13 to P-529; L-14 to P-529; S-15 to P-529; E-16to P-529; V-17 to P-529; A-18 to P-529; N-19 to P-529; G-20 to P-529;L-21 to P-529; R-22 to P-529; N-23 to P-529; P-24 to P-529; V-25 toP-529; S-26 to P-529; M-27 to P-529; V-28 to P-529; H-29 to P-529; A-30to P-529; G-31 to P-529; D-32 to P-529; G-33 to P-529; T-34 to P-529;H-35 to P-529; R-36 to P-529; F-37 to P-529; F-38 to P-529; V-39 toP-529; A-40 to P-529; E-41 to P-529; Q-42 to P-529; V-43 to P-529; G-44to P-529; V-45 to P-529; V-46 to P-529; W-47 to P-529; V-48 to P-529;Y-49 to P-529; L-50 to P-529; P-51 to P-529; D-52 to P-529; G-53 toP-529; S-54 to P-529; R-55 to P-529; L-56 to P-529; E-57 to P-529; Q-58to P-529; P-59 to P-529; F-60 to P-529; L-61 to P-529; D-62 to P-529;L-63 to P-529; K-64 to P-529; N-65 to P-529; I-66 to P-529; V-67 toP-529; L-68 to P-529; T-69 to P-529; T-70 to P-529; P-71 to P-529; W-72to P-529; I-73 to P-529; G-74 to P-529; D-75 to P-529; E-76 to P-529;R-77 to P-529; G-78 to P-529; F-79 to P-529; L-80 to P-529; G-81 toP-529; L-82 to P-529; A-83 to P-529; F-84 to P-529; H-85 to P-529; P-86to P-529; K-87 to P-529; F-88 to P-529; R-89 to P-529; H-90 to P-529;N-91 to P-529; R-92 to P-529; K-93 to P-529; F-94 to P-529; Y-95 toP-529; I-96 to P-529; Y-97 to P-529; Y-98 to P-529; S-99 to P-529; C-100to P-529; L-101 to P-529; D-102 to P-529; K-103 to P-529; K-104 toP-529; K-105 to P-529; V-106 to P-529; E-107 to P-529; K-108 to P-529;I-109 to P-529; R-110 to P-529; I-111 to P-529; S-112 to P-529; E-113 toP-529; M-114 to P-529; K-115 to P-529; V-116 to P-529; S-117 to P-529;R-118 to P-529; A-119 to P-529; D-120 to P-529; P-121 to P-529; N-122 toP-529; K-123 to P-529; A-124 to P-529; D-125 to P-529; L-126 to P-529;K-127 to P-529; S-128 to P-529; E-129 to P-529; R-130 to P-529; V-131 toP-529; I-132 to P-529; L-133 to P-529; E-134 to P-529; I-135 to P-529;E-136 to P-529; E-137 to P-529; P-138 to P-529; A-139 to P-529; S-140 toP-529; N-141 to P-529; H-142 to P-529; N-143 to P-529; G-144 to P-529;G-145 to P-529; Q-146 to P-529; L-147 to P-529; L-148 to P-529; F-149 toP-529; G-150 to P-529; L-151 to P-529; D-152 to P-529; G-153 to P-529;Y-154 to P-529; M-155 to P-529; Y-156 to P-529; I-157 to P-529; F-158 toP-529; T-159 to P-529; G-160 to P-529; D-161 to P-529; G-162 to P-529;G-163 to P-529; Q-164 to P-529; A-165 to P-529; G-166 to P-529; D-167 toP-529; P-168 to P-529; F-169 to P-529; G-170 to P-529; L-171 to P-529;F-172 to P-529; G-173 to P-529; N-174 to P-529; A-175 to P-529; Q-176 toP-529; N-177 to P-529; K-178 to P-529; S-179 to P-529; S-180 to P-529;L-181 to P-529; L-182 to P-529; G-183 to P-529; K-184 to P-529; V-185 toP-529; L-186 to P-529; R-187 to P-529; I-188 to P-529; D-189 to P-529;V-190 to P-529; N-191 to P-529; R-192 to P-529; A-193 to P-529; G-194 toP-529; S-195 to P-529; H-196 to P-529; G-197 to P-529; K-198 to P-529;R-199 to P-529; Y-200 to P-529; R-201 to P-529; V-202 to P-529; P-203 toP-529; S-204 to P-529; D-205 to P-529; N-206 to P-529; P-207 to P-529;F-208 to P-529; V-209 to P-529; S-210 to P-529; E-211 to P-529; P-212 toP-529; G-213 to P-529; A-214 to P-529; H-215 to P-529; P-216 to P-529;A-217 to P-529; I-218 to P-529; Y-219 to P-529; A-220 to P-529; Y-221 toP-529; G-222 to P-529; I-223 to P-529; R-224 to P-529; N-225 to P-529;M-226 to P-529; W-227 to P-529; R-228 to P-529; C-229 to P-529; A-230 toP-529; V-231 to P-529; D-232 to P-529; R-233 to P-529; G-234 to P-529;D-235 to P-529; P-236 to P-529; I-237 to P-529; T-238 to P-529; R-239 toP-529; Q-240 to P-529; G-241 to P-529; R-242 to P-529; G-243 to P-529;R-244 to P-529; I-245 to P-529; F-246 to P-529; C-247 to P-529; G-248 toP-529; D-249 to P-529; V-250 to P-529; G-251 to P-529; Q-252 to P-529;N-253 to P-529; R-254 to P-529; F-255 to P-529; E-256 to P-529; E-257 toP-529; V-258 to P-529; D-259 to P-529; L-260 to P-529; I-261 to P-529;L-262 to P-529; K-263 to P-529; G-264 to P-529; G-265 to P-529; N-266 toP-529; Y-267 to P-529; G-268 to P-529; W-269 to P-529; R-270 to P-529;A-271 to P-529; K-272 to P-529; E-273 to P-529; G-274 to P-529; F-275 toP-529; A-276 to P-529; C-277 to P-529; Y-278 to P-529; D-279 to P-529;K-280 to P-529; K-281 to P-529; L-282 to P-529; C-283 to P-529; H-284 toP-529; N-285 to P-529; A-286 to P-529; S-287 to P-529; L-288 to P-529;D-289 to P-529; D-290 to P-529; V-291 to P-529; L-292 to P-529; P-293 toP-529; I-294 to P-529; Y-295 to P-529; A-296 to P-529; Y-297 to P-529;G-298 to P-529; H-299 to P-529; A-300 to P-529; V-301 to P-529; G-302 toP-529; K-303 to P-529; S-304 to P-529; V-305 to P-529; T-306 to P-529;G-307 to P-529; G-308 to P-529; Y-309 to P-529; V-310 to P-529; Y-311 toP-529; R-312 to P-529; G-313 to P-529; C-314 to P-529; E-315 to P-529;S-316 to P-529; P-317 to P-529; N-318 to P-529; L-319 to P-529; N-320 toP-529; G-321 to P-529; L-322 to P-529; Y-323 to P-529; I-324 to P-529;F-325 to P-529; G-326 to P-529; D-327 to P-529; F-328 to P-529; M-329 toP-529; S-330 to P-529; G-331 to P-529; R-332 to P-529; L-333 to P-529;M-334 to P-529; A-335 to P-529; L-336 to P-529; Q-337 to P-529; E-338 toP-529; D-339 to P-529; R-340 to P-529; K-341 to P-529; N-342 to P-529;K-343 to P-529; K-344 to P-529; W-345 to P-529; K-346 to P-529; K-347 toP-529; Q-348 to P-529; D-349 to P-529; L-350 to P-529; C-351 to P-529;L-352 to P-529; G-353 to P-529; S-354 to P-529; T-355 to P-529; T-356 toP-529; S-357 to P-529; C-358 to P-529; A-359 to P-529; F-360 to P-529;P-361 to P-529; G-362 to P-529; L-363 to P-529; I-364 to P-529; S-365 toP-529; T-366 to P-529; H-367 to P-529; S-368 to P-529; K-369 to P-529;F-370 to P-529; I-371 to P-529; I-372 to P-529; S-373 to P-529; F-374 toP-529; A-375 to P-529; E-376 to P-529; D-377 to P-529; E-378 to P-529;A-379 to P-529; G-380 to P-529; E-381 to P-529; L-382 to P-529; Y-383 toP-529; F-384 to P-529; L-385 to P-529; A-386 to P-529; T-387 to P-529;S-388 to P-529; Y-389 to P-529; P-390 to P-529; S-391 to P-529; A-392 toP-529; Y-393 to P-529; A-394 to P-529; P-395 to P-529; R-396 to P-529;G-397 to P-529; S-398 to P-529; I-399 to P-529; Y-400 to P-529; K-401 toP-529; F-402 to P-529; V-403 to P-529; D-404 to P-529; P-405 to P-529;S-406 to P-529; R-407 to P-529; R-408 to P-529; A-409 to P-529; P-410 toP-529; P-411 to P-529; G-412 to P-529; K-413 to P-529; C-414 to P-529;K-415 to P-529; Y-416 to P-529; K-417 to P-529; P-418 to P-529; V-419 toP-529; P-420 to P-529; V-421 to P-529; R-422 to P-529; T-423 to P-529;K-424 to P-529; S-425 to P-529; K-426 to P-529; R-427 to P-529; I-428 toP-529; P-429 to P-529; F-430 to P-529; R-431 to P-529; P-432 to P-529;L-433 to P-529; A-434 to P-529; K-435 to P-529; T-436 to P-529; V-437 toP-529; L-438 to P-529; D-439 to P-529; L-440 to P-529; L-441 to P-529;K-442 to P-529; E-443 to P-529; Q-444 to P-529; S-445 to P-529; E-446 toP-529; K-447 to P-529; A-448 to P-529; A-449 to P-529; R-450 to P-529;K-451 to P-529; S-452 to P-529; S-453 to P-529; S-454 to P-529; A-455 toP-529; T-456 to P-529; L-457 to P-529; A-458 to P-529; S-459 to P-529;G-460 to P-529; P-461 to P-529; A-462 to P-529; Q-463 to P-529; G-464 toP-529; L-465 to P-529; S-466 to P-529; E-467 to P-529; K-468 to P-529;G-469 to P-529; S-470 to P-529; S-471 to P-529; K-472 to P-529; K-473 toP-529; L-474 to P-529; A-475 to P-529; S-476 to P-529; P-477 to P-529;T-478 to P-529; S-479 to P-529; S-480 to P-529; K-481 to P-529; N-482 toP-529; T-483 to P-529; L-484 to P-529; R-485 to P-529; G-486 to P-529;P-487 to P-529; G-488 to P-529; T-489 to P-529; K-490 to P-529; K-491 toP-529; K-492 to P-529; A-493 to P-529; R-494 to P-529; V-495 to P-529;G-496 to P-529; P-497 to P-529; H-498 to P-529; V-499 to P-529; R-500 toP-529; Q-501 to P-529; G-502 to P-529; K-503 to P-529; R-504 to P-529;R-505 to P-529; K-506 to P-529; S-507 to P-529; L-508 to P-529; K-509 toP-529; S-510 to P-529; H-511 to P-529; S-512 to P-529; G-513 to P-529;R-514 to P-529; M-515 to P-529; R-516 to P-529; P-517 to P-529; S-518 toP-529; A-519 to P-529; E-520 to P-529; Q-521 to P-529; K-522 to P-529;R-523 to P-529; A-524 to P-529; of SEQ ID NO:29. Polynucleotidesencoding these polypeptides are also encompassed by the invention, asare antibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g., fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities (e.g., ability to illicit mitogenicactivity, induce differentiation of normal or malignant cells, bind toEGF receptors, etc.)), may still be retained. For example the ability toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptide generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a mutein with alarge number of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the polypeptide shown in FIGS. 1A-C, up to the glutamineresidue at position number 7, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m1 of FIGS. 1A-C, wherem1 is an integer from 7 to 528 corresponding to the position of theamino acid residue in FIGS. 1A-C. Moreover, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of C-terminal deletions of thepolypeptide of the invention shown as SEQ ID NO:29 include polypeptidescomprising the amino acid sequence of residues: M-1 to L-528; M-1 toS-527; M-1 to R-526; M-1 to G-525; M-1 to A-524; M-1 to R-523; M-1 toK-522; M-1 to Q-521; M-1 to E-520; M-1 to A-519; M-1 S-518; M-1 toP-517; M-1 to R-516; M-1 to M-515; M-1 to R-514; M-1 to G-513; M-1 toS-512; M-1 to H-511; M-1 to S-510; M-1 to K-509; M-1 to L-508; M-1 toS-507; M-1 to K-506; M-1 to R-505; M-1 to R-504; M-1 to K-503; M-1 toG-502; M-1 to Q-501; M-1 to R-500; M-1 to V-499; M-1 to H-498; M-1 toP-497; M-1 to G-496; M-1 to V-495; M-1 to R-494; M-1 to A-493; M-1 toK-492; M-1 to K-491; M-1 to K-490; M-1 to T-489; M-1 to G-488; M-1 toP-487; M-1 to G-486; M-1 to R-485; M-1 to L-484; M-1 to T-483; M-1 toN-482; M-1 to K-481; M-1 to S-480; M-1 to S-479; M-1 to T-478; M-1 toP-477; M-1 to S-476; M-1 to A-475; M-1 to L-474; M-1 to K-473; M-1 toK-472; M-1 to S-471; M-1 to S-470; M-1 to G-469; M-1 to K-468; M-1 toE-467; M-1 to S-466; M-1 to L-465; M-1 to G-464; M-1 to Q-463; M-1 toA-462; M-1 to P-461; M-1 to G-460; M-1 to S-459; M-1 to A-458; M-1 toL-457; M-1 to T-456; M-1 to A-455; M-1 to S-454; M-1 to S-453; M-1 toS-452; M-1 to K-451; M-1 to R-450; M-1 to A-449; M-1 to A-448; M-1 toK-447; M-1 to E-446; M-1 to S-445; M-1 to Q-444; M-1 to E-443; M-1 toK-442; M-1 to L-441; M-1 to L-440; M-1 to D-439; M-1 to L-438; M-1 toV-437; M-1 to T-436; M-1 to K-435; M-1 to A-434; M-1 to L-433; M-1 toP-432; M-1 to R-431; M-1 to F-430; M-1 to P-429; M-1 to I-428; M-1 toR-427; M-1 to K-426; M-1 to S-425; M-1 to K-424; M-1 to T-423; M-1 toR-422; M-1 to V-421; M-1 to P-420; M-1 to V-419; M-1 to P-418; M-1 toK-417; M-1 to Y-416; M-1 to K-415; M-1 to C-414; M-1 to K-413; M-1 toG-412; M-1 to P-411; M-1 to P-410; M-1 to A-409; M-1 to R-408; M-1 toR-407; M-1 to S-406; M-1 to P-405; M-1 to D-404; M-1 to V-403; M-1 toF-402; M-1 to K-401; M-1 to Y-400; M-1 to I-399; M-1 to S-398; M-1 toG-397; M-1 to R-396; M-1 to P-395; M-1 to A-394; M-1 to Y-393; M-1 toA-392; M-1 to S-391; M-1 to P-390; M-1 to Y-389; M-1 to S-388; M-1 toT-387; M-1 to A-386; M-1 to L-385; M-1 to F-384; M-1 to Y-383; M-1 toL-382; M-1 to E-381; M-1 to G-380; M-1 to A-379; M-1 to E-378; M-1 toD-377; M-1 to E-376; M-1 to A-375; M-1 to F-374; M-1 to S-373; M-1 toI-372; M-1 to I-371; M-1 to F-370; M-1 to K-369; M-1 to S-368; M-1 toH-367; M-1 to T-366; M-1 to S-365; M-1 to I-364; M-1 to L-363; M-1 toG-362; M-1 to P-361; M-1 to F-360; M-1 to A-359; M-1 to C-358; M-1 toS-357; M-1 to T-356; M-1 to T-355; M-1 to S-354; M-1 to G-353; M-1 toL-352; M-1 to C-351; M-1 to L-350; M-1 to D-349; M-1 to Q-348; M-1 toK-347; M-1 to K-346; M-1 to W-345; M-1 to K-344; M-1 to K-343; M-1 toN-342; M-1 to K-341; M-1 to R-340; M-1 to D-339; M-1 to E-338; M-1 toQ-337; M-1 to L-336; M-1 to A-335; M-1 to M-334; M-1 to L-333; M-1 toR-332; M-1 to G-331; M-1 to S-330; M-1 to M-329; M-1 to F-328; M-1 toD-327; M-1 to G-326; M-1 to F-325; M-1 to I-324; M-1 to Y-323; M-1 toL-322; M-1 to G-321; M-1 to N-320; M-1 to L-319; M-1 to N-318; M-1 toP-317; M-1 to S-316; M-1 to E-315; M-1 to C-314; M-1 to G-313; M-1 toR-312; M-1 to Y-311; M-1 to V-310; M-1 to Y-309; M-1 to G-308; M-1 toG-307; M-1 to T-306; M-1 to V-305; M-1 to S-304; M-1 to K-303; M-1 toG-302; M-1 to V-301; M-1 to A-300; M-1 to H-299; M-1 to G-298; M-1 toY-297; M-1 to A-296; M-1 to Y-295; M-1 to I-294; M-1 to P-293; M-1 toL-292; M-1 to V-291; M-1 to D-290; M-1 to D-289; M-1 to L-288; M-1 toS-287; M-1 to A-286; M-1 to N-285; M-1 to H-284; M-1 to C-283; M-1 toL-282; M-1 to K-281; M-1 to K-280; M-1 to D-279; M-1 to Y-278; M-1 toC-277; M-1 to A-276; M-1 to F-275; M-1 to G-274; M-1 to E-273; M-1 toK-272; M-1 to A-271; M-1 to R-270; M-1 to W-269; M-1 to G-268; M-1 toY-267; M-1 to N-266; M-1 to G-265; M-1 to G-264; M-1 to K-263; M-1 toL-262; M-1 to I-261; M-1 to L-260; M-1 to D-259; M-1 to V-258; M-1 toE-257; M-1 to E-256; M-1 to F-255; M-1 to R-254; M-1 to N-253; M-1 toQ-252; M-1 to G-251; M-1 to V-250; M-1 to D-249; M-1 to G-248; M-1 toC-247; M-1 to F-246; M-1 to I-245; M-1 to R-244; M-1 to G-243; M-1 toR-242; M-1 to G-241; M-1 to Q-240; M-1 to R-239; M-1 to T-238; M-1 toI-237; M-1 to P-236; M-1 to D-235; M-1 to G-234; M-1 to R-233; M-1 toD-232; M-1 to V-231; M-1 to A-230; M-1 to C-229; M-1 to R-228; M-1 toW-227; M-1 to M-226; M-1 to N-225; M-1 to R-224; M-1 to I-223; M-1 toG-222; M-1 to Y-221; M-1 to A-220; M-1 to Y-219; M-1 to I-218; M-1 toA-217; M-1 to P-216; M-1 to H-215; M-1 to A-214; M-1 to G-213; M-1 toP-212; M-1 to E-211; M-1 to S-210; M-1 to V-209; M-1 to F-208; M-1 toP-207; M-1 to N-206; M-1 to D-205; M-1 to S-204; M-1 to P-203; M-1 toV-202; M-1 to R-201; M-1 to Y-200; M-1 to R-199; M-1 to K-198; M-1 toG-197; M-1 to H-196; M-1 to S-195; M-1 to G-194; M-1 to A-193; M-1 toR-192; M-1 to N-191; M-1 to V-190; M-1 to D-189; M-1 to I-188; M-1 toR-187; M-1 to L-186; M-1 to V-185; M-1 to K-184; M-1 to G-183; M-1 toL-182; M-1 to L-181; M-1 to S-180; M-1 to S-179; M-1 to K-178; M-1 toN-177; M-1 to Q-176; M-1 to A-175; M-1 to N-174; M-1 to G-173; M-1 toF-172; M-1 to L-171; M-1 to G-170; M-1 to F-169; M-1 to P-168; M-1 toD-167; M-1 to G-166; M-1 to A-165; M-1 to Q-164; M-1 to G-163; M-1 toG-162; M-1 to D-161; M-1 to G-160; M-1 to T-159; M-1 to F-158; M-1 toI-157; M-1 to Y-156; M-1 to M-155; M-1 to Y-154; M-1 to G-153; M-1 toD-152; M-1 to L-151; M-1 to G-150; M-1 to F-149; M-1 to L-148; M-1 toL-147; M-1 to Q-146; M-1 to G-145; M-1 to G-144; M-1 to N-143; M-1 toH-142; M-1 to N-141; M-1 to S-140; M-1 to A-139; M-1 to P-138; M-1 toE-137; M-1 to E-136; M-1 to I-135; M-1 to E-134; M-1 to L-133; M-1 toI-132; M-1 to V-131; M-1 to R-130; M-1 to E-129; M-1 to S-128; M-1 toK-127; M-1 to L-126; M-1 to D-125; M-1 to A-124; M-1 to K-123; M-1 toN-122; M-1 to P-121; M-1 to D-120; M-1 to A-119; M-1 to R-118; M-1 toS-117; M-1 to V-116; M-1 to K-115; M-1 to M-114; M-1 to E-113; M-1 toS-112; M-1 to I-111; M-1 to R-110; M-1 to I-109; M-1 to K-108; M-1 toE-107; M-1 to V-106; M-1 to K-105; M-1 to K-104; M-1 to K-103; M-1 toD-102; M-1 to L-101; M-1 to C-100; M-1 to S-99; M-1 to Y-98; M-1 toY-97; M-1 to I-96; M-1 to Y-95; M-1 to F-94; M-1 to K-93; M-1 to R-92;M-1 to N-91; M-1 to H-90; M-1 to R-89; M-1 to F-88; M-1 to K-87; M-1 toP-86; M-1 to H-85; M-1 to F-84; M-1 to A-83; M-1 to L-82; M-1 to G-81;M-1 to L-80; M-1 to F-79; M-1 to G-78; M-1 to R-77; M-1 to E-76; M-1 toD-75; M-1 to G-74; M-1 to I-73; M-1 to W-72; M-1 to P-71; M-1 to T-70;M-1 to T-69; M-1 to L-68; M-1 to V-67; M-1 to I-66; M-1 to N-65; M-1 toK-64; M-1 to L-63; M-1 to D-62; M-1 to L-61; M-1 to F-60; M-1 to P-59;M-1 to Q-58; M-1 to E-57; M-1 to L-56; M-1 to R-55; M-1 to S-54; M-1 toG-53; M-1 to D-52; M-1 to P-51; M-1 to L-50; M-1 to Y-49; M-1 to V-48;M-1 to W-47; M-1 to V-46; M-1 to V-45; M-1 to G-44; M-1 to V-43; M-1 toQ-42; M-1 to E-41; M-1 to A-40; M-1 to V-39; M-1 to F-38; M-1 to F-37;M-1 to R-36; M-1 to H-35; M-1 to T-34; M-1 to G-33; M-1 to D-32; M-1 toG-31; M-1 to A-30; M-1 to H-29; M-1 to V-28; M-1 to M-27; M-1 to S-26;M-1 to V-25; M-1 to P-24; M-1 to N-23; M-1 to R-22; M-1 to L-21; M-1 toG-20; M-1 to N-19; M-1 to A-18; M-1 to V-17; M-1 to E-16; M-1 to S-15;M-1 to L-14; M-1 to C-13; M-1 to L-12; M-1 to Q-11; M-1 to L-10; M-1 toC-9; M-1 to G-8; M-1 to Q-7; of SEQ ID NO:29. Polynucleotides encodingthese polypeptides are also encompassed by the invention, as areantibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g., fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

Northern analysis indicates that a 2.5-3.0 kb transcript of this gene isexpressed primarily in testes tissue and A549 lung carcinoma tissue, butinterestingly is absent from normal lung tissue. This gene is alsoexpressed in osteoarthritis tissue and human fetal tissues.

Therefore, polynucleotides and polypeptides of the invention, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of diseases and conditions which include, but are not limitedto, developmental disorders, and degenerative disorders; osteoarthritis,and lung cancer. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly ofdeveloping tissues, cartilage, and bone, expression of this gene atsignificantly higher or lower levels is routinely detected in certaintissues or cell types (e.g. bone, lung, cancerous and wounded tissues)or bodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

Based upon the tissue distribution in testes tissue, lung carcinomatissue, and osteoarthritic and fetal tissues, and the homology toseveral proteins involved in the development, differentiation, and/orproliferation of various cells, polynucleotides, translation productsand antibodies corresponding to this gene may be useful for thediagnosis, detection and/or treatment of diseases and/or disordersinvolving the proliferation of cells, particularly cancers cells such aslung carcinoma cells, as well as for diseases and/or disorders of thetestes, fetal tissues and for the diagnosis, detection and/or treatmentof osteoarthritis.

The expression in lung carcinoma tissue indicates that polynucleotides,translation products and antibodies corresponding to this gene areuseful for the diagnosis, detection and/or treatment of lung carcinomas.Polynucleotides, translation products and antibodies corresponding tothis gene may be useful for the prevention of the growth of lungcarcinomas. Alternatively, polynucleotides, translation products andantibodies corresponding to this gene may be useful in the detection oflung carcinomas. Accordingly, preferred are antibodies and or smallmolecules which specifically bind a portion of the translation productof this gene. Also provided is a kit for detecting lung carcinomas. Sucha kit comprises in one embodiment an antibody specific for translationproducts corresponding to this gene bound to a solid support. Alsoprovided is a method of detecting lung carcinomas in an individual whichcomprises a step of contacting an antibody specific for translationproducts corresponding to this gene to a bodily fluid or biologicalsample from the individual, preferably serum, and ascertaining whetherantibody binds to an antigen found in the bodily fluid. Preferably theantibody is bound to a solid support and the bodily fluid is serum. Theabove embodiments, as well as other treatments and diagnostic tests(kits and methods), are more particularly described elsewhere herein.

More generally, tissue distribution in testes tissue indicates thatpolynucleotides, translation products and antibodies corresponding tothis gene are useful for the treatment and/or diagnosis of conditionsconcerning proper testicular function (e.g. endocrine function, spermmaturation), as well as cancer. Therefore, polynucleotides, translationproducts and antibodies corresponding to this gene are useful in thetreatment of male infertility and/or impotence. Polynucleotides,translation products and antibodies corresponding to this gene are alsouseful in assays designed to identify binding agents, as such agents(antagonists) are useful as male contraceptive agents. Similarly,polynucleotides, translation products and antibodies corresponding tothis gene are useful in the treatment and/or diagnosis of testicularcancer. The testes are also a site of active gene expression oftranscripts that may be expressed, particularly at low levels, in othertissues of the body. Therefore, translation products corresponding tothis gene may be expressed in other specific tissues or organs where itmay play related functional roles in other processes, such ashematopoiesis, inflammation, bone formation, and kidney function, toname a few possible target indications.

Furthermore, the tissue distribution in osteoarthritic tissue suggeststhat polynucleotides, translation products and antibodies correspondingto this gene are useful for the treatment and/or diagnosis of diseasesof connective tissue, particularly osteoarthritis, including but notlimited to inflammation, rheumatoid arthritis, and cartilage tears andphysical injury. Additionally, translation products corresponding tothis gene, as well as antibodies directed against these translationproducts, may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:11 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2595 of SEQID NO:11, b is an integer of 15 to 2609, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:11, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 2

Translation products corresponding to this gene, sometimes referred toherein as TIDE (for Ten Integrin Domains with EGF homology), sharesequence homology with integrins, which are a superfamily of dimeric abcell-surface glycoproteins that mediate the adhesive functions of manycell types, enabling cells to interact with one another and with theextracellular matrix (See Genomics 56, 169-178 (1999); all informationand references contained within this publication are hereby incorporatedherein by reference). Eight human integrin ‘b’ subunits have beendescribed to date, and in combination with the 12 known ‘a’ subunitsform a large family of heterodimeric cell surface receptors that mediatecell adhesion to counter-receptors on neighboring cells, and to ECMproteins (reviewed by Hynes, 1992). Integrin-ligand interactions arecrucial for fundamental biological processes such as cell migration andmotility, and lymphocyte extravasation.

In another embodiment, polypeptides comprising the amino acid sequenceof the open reading frame upstream of the predicted signal peptide arecontemplated by the present invention. Specifically, polypeptides of theinvention comprise, or alternatively consist of, the following aminoacid sequence: TSTPPRAVPLPKSSQAAHQRNCNSGWSPGPASLGVRGSVCPAICWWHLS

LLPPPSVNPTLQKCSSPGAAQELSMRPPGFRNFLLLASSLLFAGLSAVPQSFSPSLRSWPGAACRLSRAESERRCRAPGQPPGAALCHGRGRCDCGVCICHVTEPGMFFGPLCECHEWVCETYDGSTCAGHGKCDCGKCKCDQGWYGDACQYPTNCDLTKKKSNQMCKNSQDIICSNAGTCHCGRCKCDNSDGSGLVYGKFCECDDRECIDDETEEICGGHGKCYCGNCYCKAGWHGDKCEFQCDITPWESKRRCTSPDGKICSNRGTCVCGECTCHDVDPTGDWGDIHGDTCECDERDCRAVYDRYSDDFCSGHGQCNCGRCDCKAGWYGKKCEHPQSCTLSAEESIRKCQGSSDLPCSGRGKCECGKCTCYPPGDRRVYGKTCECDDRRCEDLDGVVCGGHGTCSCGRCVCERGWFGKLCQHPRKCNMTEEQSKNLCESADGILCSGKGSCHCGKCICSAEEWYISGEFCDCDDRDCDKHDGLICTGNGICSCGNCECWDGWNGNACEI WLGSEYP (SEQ IDNO:50). Polynucleotides encoding these polypeptides are also encompassedby the invention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

Included in this invention as preferred domains are EGF-like domainsignature 1 and 2 domains, which were identified using the ProSiteanalysis tool (Swiss Institute of Bioinformatics). A sequence of aboutthirty to forty amino-acid residues long found in the sequence ofepidermal growth factor (EGF) has been shown [1 to 6] to be present, ina more or less conserved form, in a large number of other, mostly animalproteins. The functional significance of EGF domains in what appear tobe unrelated proteins is not yet clear. However, a common feature isthat these repeats are found in the extracellular domain ofmembrane-bound proteins or in proteins known to be secreted (exception:prostaglandin G/H synthase). The EGF domain includes six cysteineresidues which have been shown (in EGF) to be involved in disulfidebonds. The main structure is a two-stranded beta-sheet followed by aloop to a C-terminal short two-stranded sheet. Subdomains between theconserved cysteines strongly vary in length as shown in the followingschematic representation of the EGF-like domain:

‘C’: conserved cysteine involved in a disulfide bond. ‘G’: oftenconserved glycine ‘a’: often conserved aromatic amino acid ‘*’: positionof both patterns. ‘x’: any residue The region between the 5th and 6thcysteine contains two conserved glycines of which at least one ispresent in most EGF-like domains. The consensus pattern is as follows:C-x-C-x(5)-G-x(2)-C [The 3 C's are involved in disulfide bonds].

Preferred polypeptides of the invention comprise, or alternativelyconsist of, an amino acid sequence selected from the group consisting ofGKCDCGKCKCDQGWYGDACQYPTNCDLTK (SEQ ID NO: 51),GGHGKCYCGNCYCKAGWHGDKCEFQCDIT (SEQ ID NO: 52),HGQCNCGRCDCKAGWYGKKCEHPQSCTLS (SEQ ID NO: 53),HGTCSCGRCVCERGWFGKLCQHPRKCNMT (SEQ ID NO: 54),GNGICSCGNCECWDGWNGNACEIWLGSEY (SEQ ID NO: 55), andICGGHGKCYCGNCYCKAGWHGDKCEFQCDITPWESK (SEQ ID NO: 73). Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Further preferred are polypeptides comprising the EGF-like domainsignature 1 and 2 domains of the sequence referenced in Table I for thisgene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additionalcontiguous amino acid residues of this referenced sequence. Theadditional contiguous amino acid residues is N-terminal or C-terminal tothe EGF-like domain signature 1 and 2 domains.

Alternatively, the additional contiguous amino acid residues is bothN-terminal and C-terminal to the EGF-like domain signature 1 and 2domains, wherein the total N- and C-terminal contiguous amino acidresidues equal the specified number. The above preferred polypeptidedomain is characteristic of a signature specific to EGF-like domain 1and 2 containing proteins. Based on the sequence similarity, thetranslation product of this gene is expected to share at least somebiological activities with EGF-like containing proteins. Such activitiesare known in the art, some of which are described elsewhere herein.

Included in this invention as preferred domains are integrins beta chaincysteine-rich domains, which were identified using the ProSite analysistool (Swiss Institute of Bioinformatics). Integrins [7,8] are a largefamily of cell surface receptors that mediate cell to cell as well ascell to matrix adhesion. Some integrins recognize the R-G-D sequence intheir extracellular matrix protein ligand. Structurally, integrinsconsist of a dimer of an alpha and a beta chain. Each subunit has alarge N-terminal extracellular domain followed by a transmembrane domainand a short C-terminal cytoplasmic region. Some receptors share a commonbeta chain while having different alpha chains. All the integrin betachains contain four repeats of a forty amino acid region in theC-terminal extremity of their extracellular domain. Each of the repeatscontains eight cysteines. The consensus pattern is as follows:C-x-[GNQ]-x(1,3)-G-x-C-x-C-x(2)-C-x-C [The five C's are probablyinvolved in disulfide bonds].

Preferred polypeptides of the invention comprise, or alternativelyconsist of, an amino acid sequence selected from the group consistingof: GQPPGAALCHGRGRCDCGVCICHVTEPGMFFGPLC (SEQ ID NO: 74),ETYDGSTCAGHGKCDCGKCKCDQGWYGDACQYP (SEQ ID NO: 58),MCKNSQDIICSNAGTCHCGRCKCDNSDGSGLVYG (SEQ ID NO: 59),IDDETEEICGGHGKCYCGNCYCKAGWHGDKC (SEQ ID NO: 60),KRRCTSPDGKICSNRGTCVCGECTCHDVDPTGDW (SEQ ID NO: 61),DRYSDDFCSGHGQCNCGRCDCKAGWYGKKCEHPQ (SEQ ID NO: 62),CQGSSDLPCSGRGKCECGKCTCYPPGDRRVYGK (SEQ ID NO: 63),CEDLDGVVCGGHGTCSCGRCVCERGWFGKLC (SEQ ID NO: 64),SADGILCSGKGSCHCGKCICSAEEWYISGEFC (SEQ ID NO: 65), andCDKHDGLICTGNGICSCGNCECWDGWNGNACEI (SEQ ID NO: 66). Polynucleotidesencoding these polypeptides are also encompassed by the invention.

Further preferred are polypeptides comprising the integrins beta chaincysteine-rich domain of the sequence referenced in Table XIV for thisgene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additionalcontiguous amino acid residues of this referenced sequence. Theadditional contiguous amino acid residues is N-terminal or C-terminal tothe integrins beta chain cysteine-rich domain.

Alternatively, the additional contiguous amino acid residues is bothN-terminal and C-terminal to the integrins beta chain cysteine-richdomain, wherein the total N- and C-terminal contiguous amino acidresidues equal the specified number. The above preferred polypeptidedomain is characteristic of a signature specific to integrin proteins.Based on the sequence similarity, the translation product of this geneis expected to share at least some biological activities with integrinproteins, and specifically those containing an integrins beta chaincysteine-rich domain. Such activities are known in the art, some ofwhich are described elsewhere herein. The following publications werereferenced above and are hereby incorporated herein by reference: [1]Davis C. G., New Biol. 2:410-419(1990); [2] Blomquist M. C., Hunt L. T.,Barker W. C., Proc. Natl. Acad. Sci. U.S.A. 81:7363-7367(1984); [3]Barker W. C., Johnson G. C., Hunt L. T., George D. G., Protein Nucl.Acid Enz. 29:54-68(1986); [4] Doolittle R. F., Feng D. F., Johnson M.S., Nature 307:558-560(1984); [5] Appella E., Weber I. T., Blasi F.,FEBS Lett. 231:1-4(1988); [6] Campbell I. D., Bork P., Curr. Opin.Struct. Biol. 3:385-392(1993); [7] Hynes R. O., Cell 48:549-554(1987);and [8] Albelda S. M., Buck C. A., FASEB J. 4:2868-2880(1990).

The polypeptide of the present invention has been putatively identifiedas a member of the integrin family and has been termed Ten IntegrinDomains with EGF homology (“TIDE”). This identification has been made asa result of amino acid sequence homology to the human integrin beta-8subunit (See Genbank Accession No. gi|184521).

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, or all nineteen of the immunogenicepitopes shown in SEQ ID NO: 30 as residues: Met-1 to Phe-6, Arg-44 toArg-52, His-64 to Cys-69, Tyr-99 to Gln-147, His-158 to Gly-169, Phe-177to Asp-182, Cys-194 to Cys-202, Gly-213 to Phe-218, Pro-224 to Gly-236,Asp-254 to Trp-261, Asp-263 to Ala-303, Trp-305 to Cys-316, Lys-326 toAsp-332, Pro-334 to Cys-343, Pro-350 to Asp-370, Thr-407 to Asn413,Gly-425 to Cys431, Asp-449 to Asp-459, and/or Gly472 to Asn-483.Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

FIGS. 4A-C shows the nucleotide (SEQ ID NO:12) and deduced amino acidsequence (SEQ ID NO:30) of TIDE. Predicted amino acids from about 1 toabout 23 constitute the predicted signal peptide (amino acid residuesfrom about 1 to about 23 in SEQ ID NO:30) and are represented by theunderlined amino acid regions; amino acids from about 108 to about 136,from about 195 to about 223, from about 291 to about 319, from about 379to about 407, and/or from about 465 to about 493 constitute thepredicted EGF-like domain signature 1 and 2 domains (amino acids fromabout 108 to about 136, from about 195 to about 223, from about 291 toabout 319, from about 379 to about 407, and/or from about 465 to about493 in SEQ ID NO:30) and are represented by the double underlined aminoacids; and amino acids from about 55 to about 89, from about 97 to about129, from about 142 to about 175, from about 186 to about 216, fromabout 228 to about 261, from about 281 to about 314, from about 327 toabout 359, from about 368 to about 398, from about 417 to about 448,and/or from about 455 to about 487 constitute the predicted integrinsbeta chain cysteine-rich domains (amino acids from about 55 to about 89,from about 97 to about 129, from about 142 to about 175, from about 186to about 216, from about 228 to about 261, from about 281 to about 314,from about 327 to about 359, from about 368 to about 398, from about 417to about 448, and/or from about 455 to about 487 in SEQ ID NO:30) andare represented by the shaded amino acids.

FIG. 5 shows the regions of similarity between the amino acid sequencesof the Ten Integrin Domains with EGF homology (TIDE) protein (SEQ IDNO:30) and the human integrin beta-8 subunit (SEQ ID NO: 67).

FIG. 6 shows an analysis of the Ten Integrin Domains with EGF homology(TIDE) amino acid sequence. Alpha, beta, turn and coil regions;hydrophilicity and hydrophobicity; amphipathic regions; flexibleregions; antigenic index and surface probability are shown.

Translation products corresponding to this gene share homology to thecharacteristic integrins beta chain cysteine-rich domains of integrinfamily members. The polynucleotide contains an open reading frameencoding the TIDE polypeptide of 494 amino acids. TIDE exhibits a highdegree of homology at the amino acid level to the human integrin beta-8subunit (as shown in FIG. 5).

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the TIDE polypeptide having theamino acid sequence shown in FIGS. 4A-C (SEQ ID NO:30). The nucleotidesequence shown in FIGS. 4A-C (SEQ ID NO:12) was obtained by sequencing acloned cDNA (HOHCH55), which was deposited on November 17 at theAmerican Type Culture Collection, and given Accession Number 203484. Thepresent invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO:12 is intended DNA fragments atleast about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:12. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:12. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Representative examples of TIDE polynucleotide fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, from about 501 toabout 550, from about 551 to about 600, from about 601 to about 650,from about 651 to about 700, from about 701 to about 750, from about 751to about 800, from about 801 to about 850, from about 851 to about 900,from about 901 to about 950, from about 951 to about 1000, from about1001 to about 1050, from about 1051 to about 1100, from about 1101 toabout 1150, from about 1151 to about 1200, from about 1201 to about1250, from about 1251 to about 1300, from about 1301 to about 1350, fromabout 1351 to about 1400, from about 1401 to about 1450, from about 1451to about 1500, from about 1501 to about 1550, from about 1551 to about1600, from about 1601 to about 1650, from about 1651 to about 1700, fromabout 1701 to about 1750, from about 1751 to about 1800, from about 1801to about 1850, from about 1851 to about 1900, from about 1901 to about1950, from about 1951 to about 2000, from about 2001 to about 2050, fromabout 2051 to about 2100, from about 2101 to about 2150, from about 2151to about 2200, from about 2201 to about 2250, from about 2251 to about2300, from about 2301 to about 2350, from about 2351 to about 2400, fromabout 2401 to about 2450, from about 2451 to about 2499, from about 289to about 1705, and/or from about 221 to about 1705 of SEQ ID NO:12, orthe complementary strand thereto, or the cDNA contained in the depositedgene. In this context “about” includes the particularly recited ranges,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding a member selected from the group: apolypeptide comprising or alternatively, consisting of, the mature TIDEprotein (amino acid residues from about 221 to about 1705 in FIGS. 4AC(amino acids from about 221 to about 1705 in SEQ ID NO:30). Since thelocation of these domains have been predicted by computer analysis, oneof ordinary skill would appreciate that the amino acid residuesconstituting these domains may vary slightly (e.g., by about 1 to 15amino acid residues) depending on the criteria used to define eachdomain. In additional embodiments, the polynucleotides of the inventionencode functional attributes of TIDE.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of TIDE. The data representing the structural orfunctional attributes of TIDE set forth in FIG. 6 and/or Table II, asdescribed above, was generated using the various modules and algorithmsof the DNA*STAR set on default parameters. In a preferred embodiment,the data presented in columns VIII, IX XIII, and XIV of Table II can beused to determine regions of TIDE which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX XIII, and/or XIV by choosingvalues which represent regions of the polypeptide which are likely to beexposed on the surface of the polypeptide in an environment in whichantigen recognition may occur in the process of initiation of an immuneresponse.

Certain preferred regions in these regards are set out in FIG. 6, butmay, as shown in Table II, be represented or identified by using tabularrepresentations of the data presented in FIG. 6. The DNA*STAR computeralgorithm used to generate FIG. 6 (set on the original defaultparameters) was used to present the data in FIG. 6 in a tabular format(See Table II). The tabular format of the data in FIG. 6 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 6 and in Table IIinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS. 4A-C.As set out in FIG. 6 and in Table II, such preferred regions includeGarnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, etc.) may still beretained. For example, the ability of shortened TIDE muteins to induceand/or bind to antibodies which recognize the complete or mature formsof the polypeptides generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the N-terminus. Whether a particular polypeptide lackingN-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that an TIDE muteinwith a large number of deleted N-terminal amino acid residues may retainsome biological or immunogenic activities. In fact, peptides composed ofas few as six TIDE amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the TIDE aminoacid sequence shown in FIGS. 4A-C, up to the leucine residue at positionnumber 489 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-494 of FIGS. 4A-C, where n1 is aninteger from 2 to 489 corresponding to the position of the amino acidresidue in FIGS. 4A-C (which is identical to the sequence shown as SEQID NO:30). In another embodiment, N-terminal deletions of the TIDEpolypeptide can be described by the general formula n2-494, where n2 isa number from 2 to 489, corresponding to the position of amino acididentified in FIGS. 4A-C. N-terminal deletions of the TIDE polypeptideof the invention shown as SEQ ID NO:30 include polypeptides comprisingthe amino acid sequence of residues: N-terminal deletions of the TIDEpolypeptide of the invention shown as SEQ ID NO:30 include polypeptidescomprising the amino acid sequence of residues: R-2 to P494; P-3 toP-494; P-4 to P-494; G-5 to P-494; F-6 to P-494; R-7 to P-494; N-8 toP-494; F-9 to P-494; L-10 to P-494; L-11 to P-494; L-12 to P-494; A-13to P-494; S-14 to P-494; S-15 to P-494; L-16 to P-494; L-17 to P-494;F-18 to P-494; A-19 to P-494; G-20 to P-494; L-21 to P-494; S-22 toP-494; A-23 to P-494; V-24 to P-494; P-25 to P-494; Q-26 to P-494; S-27to P-494; F-28 to P-494; S-29 to P-494; P-30 to P-494; S-31 to P-494;L-32 to P-494; R-33 to P-494; S-34 to P-494; W-35 to P-494; P-36 toP-494; G-37 to P-494; A-38 to P-494; A-39 to P-494; C-40 to P-494; R-41to P-494; L-42 to P-494; S-43 to P-494; R-44 to P-494; A-45 to P-494;E-46 to P-494; S-47 to P-494; E-48 to P-494; R-49 to P-494; R-50 toP-494; C-51 to P-494; R-52 to P-494; A-53 to P-494; P-54 to P-494; G-55to P-494; Q-56 to P-494; P-57 to P-494; P-58 to P-494; G-59 to P-494;A-60 to P-494; A-61 to P-494; L-62 to P-494; C-63 to P-494; H-64 toP-494; G-65 to P-494; R-66 to P-494; G-67 to P-494; R-68 to P-494; C-69to P-494; D-70 to P-494; C-71 to P-494; G-72 to P-494; V-73 to P-494;C-74 to P-494; I-75 to P-494; C-76 to P-494; H-77 to P-494; V-78 toP-494; T-79 to P-494; E-80 to P-494; P-81 to P-494; G-82 to P-494; M-83to P-494; F-84 to P-494; F-85 to P-494; G-86 to P-494; P-87 to P-494;L-88 to P-494; C-89 to P-494; E-90 to P-494; C-91 to P-494; H-92 toP-494; E-93 to P-494; W-94 to P-494; V-95 to P-494; C-96 to P-494; E-97to P-494; T-98 to P-494; Y-99 to P-494; D-100 to P-494; G-101 to P-494;S-102 to P-494; T-103 to P-494; C-104 to P-494; A-105 to P-494; G-106 toP-494; H-107 to P-494; G-108 to P-494; K-109 to P-494; C-110 to P-494;D-111 to P-494; C-112 to P-494; G-113 to P-494; K-114 to P-494; C-115 toP-494; K-116 to P-494; C-117 to P-494; D-118 to P-494; Q-119 to P-494;G-120 to P-494; W-121 to P-494; Y-122 to P-494; G-123 to P-494; D-124 toP-494; A-125 to P-494; C-126 to P-494; Q-127 to P-494; Y-128 to P-494;P-129 to P 494; T-130 to P-494; N-131 to P-494; C-132 to P-494; D-133 toP-494; L-134 to P-494; T-135 to P-494; K-136 to P-494; K-137 to P-494;K-138 to P-494; S-139 to P-494; N-140; to P-494; Q-141 to P-494; M-142to P-494; C-143 to P-494; K-144 to P-494; N-145 to P-494; S-146 toP-494; Q-147 to P-494; D-148 to P-494; I-149 to P-494; I-150 to P-494;C-151 to P-494; S-152to P-494; N-153 to P-494; A-154 to P-494; G-155 toP-494; T-156 to P-494; C-157 to P-494; H-158 to P-494; C-159 to P-494;G-160 to P-494; R-161 to P-494; C-162 to P-494; K-163 to P-494; C-164 toP-494; D-165 to P-494; N-166 to P-494; S-167 to P-494; D-168 to P-494;G-169 to P-494; S-170 to P-494; G-171 to P-494; L-172 to P-494; V-173 toP-494; Y-174 to P-494; G-175 to P-494; K-176 to P-494; F-177 to P-494;C-178 to P-494; E-179 to P-494; C-180 to P-494; D-181 to P-494; D-182 toP-494; R-183 to P-494; E-184 to P-494; C-185 to P-494; I-186 to P-494;D-187 to P-494; D-188 to P-494; E-189 to P-494; T-190 to P-494; E-191 toP-494; E-192 to P-494; I-193 to P-494; C-194 to P-494; G-195 to P-494;G-196 to P-494; H-197 to P-494; G-198 to P-494; K-199 to P-494; C-200 toP-494; Y-201 to P-494; C-202 to P-494; G-203 to P-494; N-204 to P-494;C-205 to P-494; Y-206 to P-494; C-207 to P-494; K-208 to P-494; A-209 toP-494; G-210 to P-494; W-211 to P-494; H-212 to P-494; G-213 to P-494;D-214 to P-494; K-215 to P-494; C-216 to P-494; E-217 to P-494; F-218 toP-494; Q-219 to P-494; C-220 to P-494; D-221 to P-494; I-222 to P-494;T-223 to P-494; P-224 to P-494; W-225 to P-494; E-226 to P-494; S-227 toP-494; K-228 to P-494; R-229 to P-494; R-230 to P-494; C-231 to P-494;T-232 to P-494; S-233 to P-494; P-234 to P-494; D-235 to P-494; G-236 toP-494; K-237 to P-494; I-238 to P-494; C-239 to P-494; S-240 to P-494;N-241 to P-494; R-242 to P-494; G-243 to P-494; T-244 to P-494; C-245 toP-494; V-246 to P-494; C-247 to P-494; G-248 to P-494; E-249 to P-494;C-250 to P-494; T-251 to P-494; C-252 to P-494; H-253 to P-494; D-254 toP-494; V-255 to P-494; D-256 to P-494; P-257 to P-494; T-258 to P-494;G-259 to P-494; D-260 to P-494; W-261 to P-494; G-262 to P-494; D-263 toP-494; I-264 to P-494; H-265 to P494; G-266 to P-494; D-267 to P-494;T-268 to P-494; C-269 to P-494; E-270 to P-494; C-271 to P-494; D-272 toP-494; E-273 to P-494; R-274 to P-494; D-275 to P-494; C-276 to P-494;R-277 to P-494; A-278 to P-494; V-279 to P-494; Y-280 to P-494; D-281 toP-494; R-282 to P-494; Y-283 to P-494; S-284 to P-494; D-285 to P-494;D-286 to P-494; F-287 to P-494; C-288 to P-494; S-289 to P-494; G-290 toP-494; H-291 to P-494; G-292 to P-494; Q-293 to P-494; C-294 to P-494;N-295 to P-494; C-296 to P-494; G-297 to P-494; R-298 to P-494; C-299 toP-494; D-300 to P-494; C-301 to P-494; K-302 to P-494; A-303 to P-494;G-304 to P-494; W-305 to P-494; Y-306 to P-494; G-307 to P-494; K-308 toP-494; K-309 to P-494; C-310 to P-494; E-311 to P-494; H-312 to P-494;P-313 to P-494; Q-314 to P-494; S-315 to P-494; C-316 to P-494; T-317 toP-494; L-318 to P-494; S-319 to P-494; A-320 to P-494; E-321 to P-494;E-322 to P-494; S-323 to P-494; I-324 to P-494; R-325 to P-494; K-326 toP-494; C-327 to P-494; Q-328 to P-494; G-329 to P494; S-330 to P-494;S-331 to P-494; D-332 to P-494; L-333 to P-494; P-334 to P-494; C-335 toP-494; S-336 to P-494; G-337 to P-494; R-338 to P-494; G-339 to P-494;K-340 to P-494; C-341 to P-494; E-342 to P-494; C-343 to P-494; G-344 toP-494; K-345 to P-494; C-346 to P-494; T-347 to P-494; C-348 to P-494;Y-349 to P-494; P-350 to P-494; P-351 to P-494; G-352 to P-494; D-353 toP-494; R-354 to P-494; R-355 to P-494; V-356 to P-494; Y-357 to P-494;G-358 to P-494; K-359 to P-494; T-360 to P-494; C-361 to P-494; E-362 toP-494; C-363 to P-494; D-364 to P-494; D-365 to P-494; R-366 to P-494;R-367 to P-494; C-368 to P-494; E-369 to P-494; D-370 to P-494; L-371 toP-494; D-372 to P-494; G-373 to P-494; V-374 to P-494; V-375 to P-494;C-376 to P-494; G-377 to P-494; G-378 to P-494; H-379 to P-494; G-380 toP-494; T-381 to P-494; C-382 to P-494; S-383 to P-494; C-384 to P-494;G-385 to P-494; R-386 to P-494; C-387 to P-494; V-388 to P-494; C-389 toP-494; E-390 to P-494; R-391 to P-494; G-392 to P-494; W-393 to P-494;F-394 to P-494; G-395 to P-494; K-396 to P-494; L-397 to P-494; C-398 toP-494; Q-399 to P-494; H-400 to P-494; P-401 to P-494; R-402 to P-494;K-403 to P-494; C-404 to P-494; N-405 to P-494; M-406 to P-494; T-407 toP-494; E-408 to P-494; E-409 to P-494; Q-410 to P-494; S-411 to P-494;K-412 to P-494; N-413 to P-494; L-414 to P-494; C-415 to P-494; E-416 toP-494; S-417 to P-494; A-418 to P-494; D-419 to P-494; G-420 to P-494;I-421 to P-494; L-422 to P-494; C-423 to P-494; S-424 to P-494; G-425 toP-494; K-426 to P-494; G-427 to P-494; S-428 to P-494; C-429 to P-494;H-430 to P-494; C-431 to P-494; G-432 to P-494; K-433 to P-494; C-434 toP-494; I-435 to P-494; C-436 to P-494; S-437 to P-494; A-438 to P-494;E-439 to P-494; E-440 to P-494; W-441 to P-494; Y-442 to P-494; I-443 toP-494; S-444 to P-494; G-445 to P-494; E-446 to P-494; F-447 to P-494;C-448 to P-494; D-449 to P-494; C-450 to P-494; D-451 to P-494; D-452 toP-494; R-453 to P-494; D-454 to P-494; C-455 to P-494; D-456 to P-494;K-457 to P-494; H-458 to P-494; D-459 to P-494; G-460 to P-494; L-461 toP-494; I-462 to P-494; C-463 to P-494; T-464 to P-494; G-465 to P-494;N-466 to P-494; G-467 to P-494; I-468 to P-494; C-469 to P-494; S-470 toP-494; C-471 to P-494; G-472 to P-494; N-473 to P-494; C-474 to P-494;E-475 to P-494; C-476 to P-494; W-477 to P-494; D-478 to P-494; G-479 toP-494; W-480 to P-494; N-481 to P-494; G-482 to P-494; N-483 to P-494;A-484 to P-494; C-485 to P-494; E-486 to P-494; I-487 to P-494; W-488 toP-494; L-489 to P-494; of SEQ ID NO:30. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g., fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identicalto these polypeptides and polypeptides encoded by the polynucleotidewhich hybridizes, under stringent conditions, to the polynucleotideencoding these polypeptides, or the complement thereof) are encompassedby the invention. Antibodies that bind these fragments and variants ofthe invention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activitiesmay still be retained. For example the ability of the shortened TIDEmutein to induce and/or bind to antibodies which recognize the completeor mature forms of the polypeptide generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the C-terminus. Whether a particular polypeptidelacking C-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thatan TIDE mutein with a large number of deleted C-terminal amino acidresidues may retain some biological or immunogenic activities. In fact,peptides composed of as few as six TIDE amino acid residues may oftenevoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the TIDE polypeptide shown in FIGS. 4A-C, up to thephenylalanine residue at position number 6, and polynucleotides encodingsuch polypeptides. In particular, the present invention providespolypeptides comprising the amino acid sequence of residues 1-m1 of FIG.1, where m1 is an integer from 6 to 494 corresponding to the position ofthe amino acid residue in FIGS. 4A-C. Moreover, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of C-terminal deletions of theTIDE polypeptide of the invention shown as SEQ ID NO:30 includepolypeptides comprising the amino acid sequence of residues: M-1 toY-493; M-1 to E-492; M-1 to S-491; M-1 to G-490; M-1 to L-489; M-1 toW-488; M-1 to I-487; M-1 to E-486; M-1 to C-485; M-1 to A-484; M-1 toN-483; M-1 to G-482; M-1 to N-481; M-1 to W-480; M-1 to G-479; M-1 toD-478; M-1 to W-477; M-1 to C-476; M-1 to E-475; M-1 to C-474; M-1 toN-473; M-1 to G-472; M-1 to C-471; M-1 to S-470; M-1 to C-469; M-1 toI-468 to M-1 to G-467; M-1 to N-466; M-1 to G-465; M-1 to T-464; M-1 toC-463; M-1 to I-462; M-1 to L-461; M-1 to G-460; M-1 to D-459; M-1 toH-458; M-1 to K-457; M-1 to D-456; M-1 to C-455; M-1 to D-454; M-1 toR-453; M-1 to D-452; M-1 to D-451; M-1 to C-450; M-1 to D-449; M-1 toC-448; M-1 to F-447; M-1 to E-446; M-1 to G-445; M-1 to S-444; M-1 toI-443; M-1 to Y-442; M-1 to W-441; M-1 to E-440; M-1 to E-439; M-1 toA-438; M-1 to S-437; M-1 to C-436; M-1 to I-435; M-1 to C-434; M-1 toK-433; M-1 to G-432; M-1 to C-431; M-1 to H-430; M-1 to C-429; M-1 toS-428; M-1 to G-427; M-1 to K-426; M-1 to G-425; M-1 to S-424; M-1 toC-423; M-1 to L-422; M-1 to I-421; M-1 to G-420; M-1 to D-419; M-1 toA-418; M-1 to S-417; M-1 to E-416; M-1 to C-415; M-1 to L-414; M-1 toN-413; M-1 to K-412; M-1 to S-411; M-1 to Q-410; M-1 to E-409; M-1 toE-408; M-1 to T-407; M-1 to M-406; M-1 to N-405; M-1 to C-404; M-1 toK-403; M-1 to R-402; M-1 to P-401; M-1 to H-400; M-1 to Q-399; M-1 toC-398; M-1 to L-397; M-1 to K-396; M-1 to G-395; M-1 to F-394; M-1 toW-393; M-1 to G-392; M-1 to R-391; M-1 to E-390; M-1 to C-389; M-1 toV-388; M-1 to C-387; M-1 to R-386; M-1 to G-385; M-1 to C-384; M-1 toS-383; M-1 to C-382; M-1 to T-381; M-1 to G-380; M-1 to H-379; M-1 toG-378; M-1 to G-377; M-1 to C-376; M-1 to V-375; M-1 to V-374; M-1 toG-373; M-1 to D-372; M-1 to L-371; M-1 to D-370; M-1 to E-369; M-1 toC-368; M-1 to R-367; M-1 to R-366; M-1 to D-365; M-1 to D-364; M-1 toC-363; M-1 to E-362; M-1 to C-361; M-1 to T-360; M-1 to K-359; M-1 toG-358; M-1 to Y-357; M-1 to V-356; M-1 to R-355; M-1 to R-354; M-1 toD-353; M-1 to G-352; M-1 to P-351; M-1 to P-350; M-1 to Y-349; M-1 toC-348; M-1 to T-347; M-1 to C-346; M-1 to K-345; M-1 to G-344; M-1 toC-343; M-1 to E-342; M-1 to C-341; M-1 to K-340; M-1 to G-339; M-1 toR-338; M-1 to G-337; M-1 to S-336; M-1 to C-335; M-1 to P-334; M-1 toL-333; M-1 to D-332; M-1 to S-331; M-1 to S-330; M-1 to G-329; M-1 toQ-328; M-1 to C-327; M-1 to K-326; M-1 to R-325; M-1 to I-324; M-1 toS-323; M-1 to E-322; M-1 to E-321; M-1 to A-320; M-1 to S-319; M-1 toL-318; M-1 to T-317; M-1 to C-316; M-1 to S-315; M-1 to Q-314; M-1 toP-313; M-1 to H-312; M-1 to E-311; M-1 to C-310; M-1 to K-309; M-1 toK-308; M-1 to G-307; M-1 to Y-306; M-1 to W-305; M-1 to G-304; M-1 toA-303; M-1 to K-302; M-1 to C-301; M-1 to D-300; M-1 to C-299; M-1 toR-298; M-1 to G-297; M-1 to C-296; M-1 to N-295; M-1 to C-294; M-1 toQ-293; M-1 to G-292; M-1 to H-291; M-1 to G-290; M-1 to S-289; M-1 toC-288; M-1 to F-287; M-1 to D-286; M-1 to D-285; M-1 to S-284; M-1 toY-283; M-1 to R-282; M-1 to D-281; M-1 to Y-280; M-1 to V-279; M-1 toA-278; M-1 to R-277; M-1 to C-276; M-1 to D-275; M-1 to R-274; M-1 toE-273; M-1 to D-272; M-1 to C-271; M-1 to E-270; M-1 to C-269; M-1 toT-268; M-1 to D-267; M-1 to G-266; M-1 to H-265; M-1 to I-264; M-1 toD-263; M-1 to G-262; M-1 to W-261; M-1 to D-260; M-1 to G-259; M-1 toT-258; M-1 to P-257; M-1 to D-256; M-1 to V-255; M-1 to D-254; M-1 toH-253; M-1 to C-252; M-1 to T-251; M-1 to C-250; M-1 to E-249; M-1 toG-248; M-1 to C-247; M-1 to V-246; M-1 to C-245; M-1 to T-244; M-1 toG-243; M-1 to R-242; M-1 to N-241; M-1 to S-240; M-1 to C-239; M-1 toI-238; M-1 to K-237; M-1 to G-236; M-1 to D-235; M-1 to P-234; M-1 toS-233; M-1 to T-232; M-1 to C-231; M-1 to R-230; M-1 to R-229; M-1 toK-228; M-1 to S-227; M-1 to E-226; M-1 to W-225; M-1 to P-224; M-1 toT-223; M-1 to I-222; M-1 to D-221; M-1 to C-220; M-1 to Q-219; M-1 toF-218; M-1 to E-217; M-1 to C-216; M-1 to K-215; M-1 to D-214; M-1 toG-213; M-1 to H-212; M-1 to W-211; M-1 to G-210; M-1 to A-209; M-1 toK-208; M-1 to C-207; M-1 to Y-206; M-1 to C-205; M-1 to N-204; M-1 toG-203; M-1 to C-202; M-1 to Y-201; M-1 to C-200; M-1 to K-199; M-1 toG-198; M-1 to H-197; M-1 to G-196; M-1 to G-195; M-1 to C-194; M-1 toI-193; M-1 to E-192; M-1 to E-191; M-1 to T-190; M-1 to E-189; M-1 toD-188; M-1 to D-187; M-1 to I-186; M-1 to C-185; M-1 to E-184; M-1 toR-183; M-1 to D-182; M-1 to D-181; M-1 to C-180; M-1 to E-179; M-1 toC-178; M-1 to F-177; M-1 to K-176; M-1 to G-175; M-1 to Y-174; M-1 toV-173; M-1 to L-172; M-1 to G-171; M-1 to S-170; M-1 to G-169; M-1 toD-168; M-1 to S-167; M-1 to N-166; M-1 to D-165; M-1 to C-164; M-1 toK-163; M-1 to C-162; M-1 to R-161; M-1 to G-160; M-1 to C-159; M-1 toH-158; M-1 to C-157; M-1 to T-156; M-1 to G-155; M-1 to A-154; M-1 toN-153; M-1 to S-152; M-1 to C-151; M-1 to I-150; M-1 to I-149; M-1 toD-148; M-1 to Q-147; M-1 to S-146; M-1 to N-145; M-1 to K-144; M-1 toC-143; M-1 to M-142; M-1 to Q-141; M-1 to N-140; M-1 to S-139; M-1 toK-138; M-1 to K-137; M-1 to K-136; M-1 to T-135; M-1 to L-134; M-1 toD-133; M-1 to C-132; M-1 to N-131; M-1 to T-130; M-1 to P-129; M-1 toY-128; M-1 to Q-127; M-1 to C-126; M-1 to A-125; M-1 to D-124; M-1 toG-123; M-1 to Y-122; M-1 to W-121; M-1 to G-120; M-1 to Q-119; M-1 toD-118; M-1 to C-117; M-1 to K-116; M-1 to C-115; M-1 to K-114; M-1 toG-113; M-1 to C-112; M-1 to D-111; M-1 to C-110; M-1 to K-109; M-1 toG-108; M-1 to H-107; M-1 to G-106; M-1 to A-105; M-1 to C-104; M-1 toT-103; M-1 to S-102; M-1 to G-101; M-1 to D-100; M-1 to Y-99; M-1 toT-98; M-1 to E-97; M-1 to C-96; M-1 to V-95; M-1 to W-94; M-1 to E-93;M-1 to H-92; M-1 to C-91; M-1 to E-90; M-1 to C-89; M-1 to L-88; M-1 toP-87; M-1 to G-86; M-1 to F-85; M-1 to F-84; M-1 to M-83; M-1 to G-82;M-1 to P-81; M-1 to E-80; M-1 to T-79; M-1 to V-78; M-1 to H-77; M-1 toC-76; M-1 to I-75; M-1 to C-74; M-1 to V-73; M-1 to G-72; M-1 to C-71;M-1 to D-70; M-1 to C-69; M-1 to R-68; M-1 to G-67; M-1 to R-66; M-1 toG-65; M-1 to H-64; M-1 to C-63; M-1 to L-62; M-1 to A-61; M-1 to A-60;M-1 to G-59; M-1 to P-58; M-1 to P-57; M-1 to Q-56; M-1 to G-55; M-1 toP-54; M-1 to A-53; M-1 to R-52; M-1 to C-51; M-1 to R-50; M-1 to R-49;M-1 to E-48; M-1 to S-47; M-1 to E-46; M-1 to A-45; M-1 to R-44; M-1 toS-43; M-1 to L-42; M-1 to R-41; M-1 to C-40; M-1 to A-39; M-1 to A-38;M-1 to G-37; M-1 to P-36; M-1 to W-35; M-1 to S-34; M-1 to R-33; M-1 toL-32; M-1 to S-31; M-1 to P-30; M-1 to S-29; M-1 to F-28; M-1 to S-27;M-1 to Q-26; M-1 to P-25; M-1 to V-24; M-1 to A-23; M-1 to S-22; M-1 toL-21; M-1 to G-20; M-1 to A-19; M-1 to F-18; M-1 to L-17; M-1 to L-16;M-1 to S-15; M-1 to S-14; M-1 to A-13; M-1 to L-12; M-1 to L-11; M-1 toL-10; M-1 to F-9; M-1 to N-8; M-1 to R-7; M-1 to F-6; of SEQ ID NO:30.Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 990% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybrdizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:12 whichhave been determined from the following related cDNA genes: HLHFV34R(SEQ ID NO:68), HSRDA85R (SEQ ID NO:69), HSRAZ62R (SEQ ID NO:70),HSRDA17R (SEQ ID NO:71), and HSLEC45R (SEQ ID NO:72).

A polynucleotide encoding a polypeptide of the present invention isobtained from human osteoblasts, synovial hypoxia tissue, osteoblast andosteoclast, bone marrow stromal cells, umbilical vein, smooth muscle,placenta, and fetal lung. The polynucleotide of this invention wasdiscovered in a human osteoblast II cDNA library.

This gene is expressed primarily in synovial hypoxia tissue, osteoblastand osteoclast, bone marrow stromal cells, and to a lesser extent inumbilical vein, smooth muscle, placenta, and fetal lung cDNA libraries.Therefore, polynucleotides and polypeptides of the invention, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of diseases and conditions which include, but are not limitedto, disorders of bone and connective tissues, immune and hematopoieticdiseases and/or disorders, vascular disorders, and other disordersinvolving aberrations in cell-surface interactions. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the connective tissue and skeletalsystem, expression of this gene at significantly higher or lower levelsis routinely detected in certain tissues or cell types (e.g., cartilage,bone, vascular, hypoxic tissue, and cancerous and wounded tissues) orbodily fluids (e.g., lymph, serum, plasma, urine, synovial fluid andspinal fluid) or another tissue or cell sample taken from an individualhaving such a disorder, relative to the standard gene expression level,i.e., the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

Based on the sequence similarity to the human integrin beta-8 subunit,translation product of this gene is expected to share at least somebiological activities with integrin proteins, and specifically the humanintegrin beta-8 subunit. Such activities are known in the art, some ofwhich are described elsewhere herein.

Specifically, polynucleotides, translation products and antibodiescorresponding to this gene are also useful for modulating thedifferentiation of normal and malignant cells, modulating theproliferation and/or differentiation of cancer and neoplastic cells, andmodulating the immune response. Polynucleotides and polypeptides of theinvention may represent a diagnostic marker for hematopoietic and immunediseases and/or disorders. The full-length protein should be a secretedprotein, based upon homology to the integrin family. Therefore, it issecreted into serum, urine, or feces and thus the levels is assayablefrom patient samples. Assuming specific expression levels are reflectiveof the presence of immune disorders, this protein would provide aconvenient diagnostic for early detection. In addition, expression ofthis gene product may also be linked to the progression of immunediseases, and therefore may itself actually represent a therapeutic ortherapeutic target for the treatment of cancer. Polynucleotides andpolypeptides of the invention may play an important role in thepathogenesis of human cancers and cellular transformation, particularlythose of the immune and hematopoietic systems. Polynucleotides andpolypeptides of the invention may also be involved in the pathogenesisof developmental abnormalities based upon its potential effects onproliferation and differentiation of cells and tissue cell types. Due tothe potential proliferating and differentiating activity of saidpolynucleotides and polypeptides, the invention is useful as atherapeutic agent in inducing tissue regeneration, for treatinginflammatory conditions (e.g., inflammatory bowel syndrome,diverticulitis, etc.). Moreover, the invention is useful in modulatingthe immune response to aberrant polypeptides, as may exist in rapidlyproliferating cells and tissue cell types, particularly inadenocarcinoma cells, and other cancers.

Alternatively, the expression within cellular sources marked byproliferating cells indicates polynucleotides, translation products andantibodies corresponding to this gene may play a role in the regulationof cellular division, and may show utility in the diagnosis, treatment,and/or prevention of developmental diseases and disorders, includingcancer, and other proliferative conditions. Representative uses aredescribed in the “Hyperproliferative Disorders” and “Regeneration”sections below and elsewhere herein. Briefly, developmental tissues relyon decisions involving cell differentiation and/or apoptosis in patternformation.

Dysregulation of apoptosis can result in inappropriate suppression ofcell death, as occurs in the development of some cancers, or in failureto control the extent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA).

Alternatively, this gene product is involved in the pattern of cellularproliferation that accompanies early embryogenesis. Thus, aberrantexpression of this gene product in tissues—particularly adulttissues—may correlate with patterns of abnormal cellular proliferation,such as found in various cancers. Because of potential roles inproliferation and differentiation, polynucleotides, translation productsand antibodies corresponding to this gene may have applications in theadult for tissue regeneration and the treatment of cancers. It may alsoact as a morphogen to control cell and tissue type specification.Therefore, polynucleotides, translation products and antibodiescorresponding to this gene are useful in treating, detecting, and/orpreventing said disorders and conditions, in addition to other types ofdegenerative conditions. Thus this protein may modulate apoptosis ortissue differentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases. Theprotein is useful in modulating the immune response to aberrantpolypeptides, as may exist in proliferating and cancerous cells andtissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

The translation product of this gene, sometimes referred to herein asTIDE (for Ten Integrin Domains with EGF homology), shares sequencehomology with integrins, which are a superfamily of dimeric abcell-surface glycoproteins that mediate the adhesive functions of manycell types, enabling cells to interact with one another and with theextracellular matrix (See Genomics 56, 169-178 (1999); all informationand references contained within this publication are hereby incorporatedherein by reference).

The gene encoding the disclosed cDNA is believed to reside on chromosome13, at locus 13q33. Accordingly, polynucleotides related to thisinvention are useful as a marker in linkage analysis for chromosome 13,generally, and particularly at locus 13q33.

The tissue distribution and homology to the human integrin beta-8subunit indicates polynucleotides and polypeptides corresponding to thisgene are useful for the diagnosis and treatment of a variety of immunesystem disorders. Representative uses are described in the “ImmuneActivity” and “infectious disease” sections below, in Example 11, 13,14, 16, 18, 19, 20, and 27, and elsewhere herein.

Briefly, the expression indicates a role in regulating theproliferation; survival; differentiation; and/or activation ofhematopoietic cell lineages, including blood stem cells. Involvement inthe regulation of cytokine production antigen presentation, or otherprocesses indicates a usefulness for treatment of cancer (e.g. byboosting immune responses). Expression in cells of lymphoid origin,indicates the natural gene product is involved in immune functions.Therefore it would also be useful as an agent for immunologicaldisorders including arthritis, asthma, immunodeficiency diseases such asAIDS, leukemia, rheumatoid arthritis, granulomatous Disease,inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia,psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity;immune reactions to transplanted organs and tissues, such ashost-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's Disease, and scleroderma.Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Based upon the tissue distributionof this protein, antagonists directed against this protein is useful inblocking the activity of this protein. Accordingly, preferred areantibodies which specifically bind a portion of the translation productof this gene.

Also provided is a kit for detecting tumors in which expression of thisprotein occurs. Such a kit comprises in one embodiment an antibodyspecific for the translation product of this gene bound to a solidsupport. Also provided is a method of detecting these tumors in anindividual which comprises a step of contacting an antibody specific forthe translation product of this gene to a bodily fluid or biologicalsample from the individual, preferably serum, and ascertaining whetherantibody binds to an antigen found in the bodily fluid. Preferably theantibody is bound to a solid support and the bodily fluid is serum. Theabove embodiments, as well as other treatments and diagnostic tests(kits and methods), are more particularly described elsewhere herein.

Furthermore, the protein may also be used to determine biologicalactivity, raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:12 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2485 of SEQID NO:12, b is an integer of 15 to 2499, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:12, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 3

Translation products corresponding to this gene share sequence homologywith RAMP-3 (receptor-activity-modifying proteins), which another grouphas recently published, which is thought to be important in thetransport of the calcitonin-receptor-like receptor (CRLR) to the plasmamembrane. RAMPs regulate the transport and ligand specificity of thecalcitonin-receptor-like-receptor. There are two other relatedreceptor-activity-modifying proteins, known as RAMP1 and RAMP-2 (Nature1998 May 28;393(6683):333-9). RAMP1 is thought to present the receptorat the cell surface as a mature glycoprotein and aCalcitonin-gene-related peptide (CGRP) receptor.

Alternatively, RAMP-2-transported receptors are core-glycosylated andare adrenomedullin receptors. CGRP (a 37-amino-acid neuropeptide) andits receptors are widely distributed in the body, and it is the mostpotent endogenous vasodilatory peptide discovered so far (Crit RevNeurobiol 1997;11(2-3):167-239). Specific binding sites foradrenomedullin were present in every region of human brain (cerebralcortex, cerebellum, thalamus, hypothalamus, pons and medulla oblongata),suggesting that a novel neurotransmitter/neuromodulator role may existfor adrenomedullin in human brain (Peptides 1997;18(8):1125-9).

FIGS. 7A-B show the nucleotide (SEQ ID NO:13) and deduced amino acidsequence (SEQ ID NO:31) of the intestine derived extracellular protein.Predicted amino acids from about 1 to about 27 constitute the predictedsignal peptide (amino acid residues from about 1 to about 27 in SEQ IDNO:31) and are represented by the underlined amino acid regions; andamino acids from about 122 to about 138 constitute the predictedtransmembrane domain (amino acid residues from about 122 to about 138 inSEQ ID NO:31) and are represented by the double-underlined amino acids.

FIG. 8 shows the regions of similarity between the amino acid sequencesof the Intestine derived extracellular protein SEQ ID NO:31, and theRAMP3 protein (gi|4587099) (SEQ ID NO: 75).

FIG. 9 shows an analysis of the amino acid sequence of SEQ ID NO: 31.

Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, or all three of theimmunogenic epitopes shown in SEQ ID NO: 31 as residues: Ala-5 toGln-10, Pro-23 to Cys-28, and/or Arg-140 to Asp-145. Polynucleotidesencoding these polypeptides are also encompassed by the invention, asare antibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g., fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the polypeptide having the aminoacid sequence shown in FIG. 1 (SEQ ID NO:31), which was determined bysequencing a cloned cDNA (HTLEW81). The nucleotide sequence shown inFIGS. 7A-B (SEQ ID NO:13) was obtained by sequencing a cloned cDNA(HTLEW81), which was deposited on Nov. 17, 1998 at the American TypeCulture Collection, and given Accession Number 203484. The depositedgene is inserted in the pSport plasmid (Life Technologies, Rockville,Md.) using the SalI/NotI restriction endonuclease cleavage sites. Thepresent invention is further directed to fragments of the isolatednucleic acid molecules described herein.

By a fragment of an isolated DNA molecule having the nucleotide sequenceof the deposited cDNA or the nucleotide sequence shown in SEQ ID NO:13is intended DNA fragments at least about 15nt, and more preferably atleast about 20 nt, still more preferably at least about 30 nt, and evenmore preferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50-1500 nt in length are also useful according to the presentinvention, as are fragments corresponding to most, if not all, of thenucleotide sequence of the deposited cDNA or as shown in SEQ ID NO:13.By a fragment at least 20 nt in length, for example, is intendedfragments which include 20 or more contiguous bases from the nucleotidesequence of the deposited cDNA or the nucleotide sequence as shown inSEQ ID NO:13. In this context “about” includes the particularly recitedsize, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, ateither terminus or at both termini. Representative examples ofpolynucleotide fragments of the invention include, for example,fragments that comprise, or alternatively, consist of, a sequence fromabout nucleotide 1 to about 50, from about 51 to about 100, from about101 to about 150, from about 151 to about 200, from about 201 to about250, from about 251 to about 300, from about 301 to about 350, fromabout 351 to about 400, from about 401 to about 450, from about 451 toabout 500, and from about 501 to about 550, and from about 551 to about600, from about 601 to about 650, from about 651 to about 700, fromabout 701 to about 750, from about 751 to about 800, from about 801 toabout 850, from about 851 to about 900, from about 901 to about 950,from about 951 to about 1000, from about 1001 to about 1050, from about1051 to about 1100, from about 1101 to about 1150, from about 1151 toabout 1200, from about 1201 to about 1250, from about 1251 to about1300, from about 1301 to about 1339 of SEQ ID NO:13, or thecomplementary strand thereto, or the cDNA contained in the depositedgene. In this context “about” includes the particularly recited ranges,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. In additional embodiments, thepolynucleotides of the invention encode functional attributes of thecorresponding protein.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions. The data representing the structural or functionalattributes of the protein set forth in FIG. 9 and/or Table III, asdescribed above, was generated using the various modules and algorithmsof the DNA*STAR set on default parameters. In a preferred embodiment,the data presented in columns VIII, IX, XIII, and XIV of Table III canbe used to determine regions of the protein which exhibit a high degreeof potential for antigenicity. Regions of high antigenicity aredetermined from the data presented in columns VIII, IX, XIII, and/or XIVby choosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

Certain preferred regions in these regards are set out in FIG. 9, butmay, as shown in Table III, be represented or identified by usingtabular representations of the data presented in FIG. 9. The DNA*STARcomputer algorithm used to generate FIG. 9 (set on the original defaultparameters) was used to present the data in FIG. 9 in a tabular format(See Table III). The tabular format of the data in FIG. 9 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 9 and in Table IIIinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS. 7A-B.As set out in FIG. 9 and in Table III, such preferred regions includeGamier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, etc.) may still beretained. For example, the ability of shortened muteins to induce and/orbind to antibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a mutein with a large numberof deleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixamino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence shown in FIGS. 7A-B, up to the arginine residue at positionnumber 143 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-148 of FIGS. 7A-B, where n1 is aninteger from 2 to 143 corresponding to the position of the amino acidresidue in FIGS. 7A-B (which is identical to the sequence shown as SEQID NO:31). N-terminal deletions of the polypeptide of the inventionshown as SEQ ID NO:31 include polypeptides comprising the amino acidsequence of residues: E-2 to L-148; T-3 to L-148; G-4 to L-148; A-5 toL-148; L-6 to L-148; R-7 to L-148; R-8 to L-148; P-9 to L-148; Q-10 toL-148; L-11 to L-148; L-12 to L-148; P-13to L-148; L-14 to L-148; L-15to L-148; L-16 to L-148; L-17 to L-148; L-18 to L-148; C-19 to L-148;G-20 to L-148; G-21 to L-148; C-22 to L-148; P-23 to L-148; R-24 toL-148; A-25 to L-148; G-26 to L-148; G-27 to L-148; C-28 to L-148; N-29to L-148; E-30 to L-148; T-31 to L-148; G-32 to L-148; M-33 to L-148;L-34 to L-148; E-35 to L-148; R-36 to L-148; L-37 to L-148; P-38 toL-148; L-39 to L-148; C-40 to L-148; G-41 to L-148; K-42 to L-148; A-43to L-148; F-44 to L-148; A-45 to L-148; D-46 to L-148; M-47 to L-148;M-48 to L-148; G-49 to L-148; K-50 to L-148; V-51 to L-148; D-52 toL-148; V-53 to L-148; W-54 to L-148; K-55 to L-148; W-56 to L-148; C-57to L-148; N-58 to L-148; L-59 to L-148; S-60 to L-148; E-61 to L-148;F-62 to L-148; I-63 to L-148; V-64 to L-148; Y-65 to L-148; Y-66 toL-148; E-67 to L-148; S-68 to L-148; F-69 to L-148; T-70 to L-148; N-71to L-148; C-72 to L-148; T-73 to L-148; E-74 to L-148; M-75 to L-148;E-76 to L-148; A-77 to L-148; N-78 to L-148; V-79 to L-148; V-80 toL-148; G-81 to L-148; C-82 to L-148; Y-83 to L-148; W-84 to L-148; P-85to L-148; N-86 to L-148; P-87 to L-148; L-88 to L-148; A-89 to L-148;Q-90 to L-148; G-91 to L-148; F-92 to L-148; I-93 to L-148; T-94 toL-148; G-95 to L-148; I-96 to L-148; H-97 to L-148; R-98 to L-148; Q-99to L-148; F-100 to L-148; F-101 to L-148; S-102 to L-148; N-103 toL-148; C-104 to L-148; T-105 to L-148; V-106 to L-148; D-107 to L-148;R-108 to L-148; V-109 to L-148; H-110 to L-148; L-111 to L-148; E-112 toL-148; D-113 to L-148; P-114 to L-148; P-115 to L-148; D-116 to L-148;E-117 to L-148; V-118 to L-148; L-119 to L-148; I-120 to L-148; P-121 toL-148; L-122 to L-148; I-123 to L-148; V-124 to L-148; I-125 to L-148;P-126 to L-148; V-127 to L-148; V-128 to L-148; L-129 to L-148; T-130 toL-148; V-131 to L-148; A-132 to L-148; M-133 to L-148; A-134 to L-148;G-135 to L-148; L-136 to L-148; V-137 to L-148; V-138 to L-148; W-139 toL-148; R-140 to L-148; S-141 to L-148; K-142 to L-148; R-143 to L-148;of SEQ ID NO:31. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities (e.g., ability to illicit mitogenicactivity, induce differentiation of normal or malignant cells, bind toEGF receptors, etc.)), may still be retained. For example the ability toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptide generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a mutein with alarge number of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the polypeptide shown in FIGS. 7A-B, up to the arginineresidue at position number 7, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m1 of FIGS. 7A-B, wherem1 is an integer from 7 to 147 corresponding to the position of theamino acid residue in FIGS. 7A-B. Moreover, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of C-terminal deletions of thepolypeptide of the invention shown as SEQ ID NO:31 include polypeptidescomprising the amino acid sequence of residues: M-1 to L-147; M-1 toT-146; M-1 to D-145; M-1 to T-144; M-1 to R-143; M-1 to K-142; M-1 toS-141; M-1 to R-140; M-1 to W-139; M-1 to V-138; M-1 to V-137; M-1 toL-136; M-1 to G-135; M-1 to A-134; M-1 to M-133; M-1 to A-132; M-1 toV-131; M-1 to T-130; M-1 to L-129; M-1 V-128; M-1 to V-127; M-1 toP-126; M-1 to I-125; M-1 to V-124; M-1 to I-123; M-1 to L-122; M-1 toP-121; M-1 to I-120; M-1 to L-119; M-1 to V-118; M-1 to E-117; M-1 toD-116; M-1 to P-115; M-1 to P-114; M-1 to D-113; M-1 to E-112; M-1 toL-111; M-1 to H-110; M-1 to V-109; M-1 to R-108; M-1 to D-107; M-1 toV-106; M-1 to T-105; M-1 to C-104; M-1 to N-103; M-1 to S-102; M-1 toF-101; M-1 to F-100; M-1 to Q-99; M-1 to R-98; M-1 to H-97; M-1 to I-96;M-1 to G-95; M-1 to T-94; M-1 to I-93; M-1 to F-92; M-1 to G-91; M-1 toQ-90; M-1 to A-89; M-1 to L-88; M-1 to P-87; M-1 to N-86; M-1 to P-85;M-1 to W-84; M-1 to Y-83; M-1 to C-82; M-1 to G-81; M-1 to V-80; M-1 toV-79; M-1 to N-78; M-1 to A-77; M-1 to E-76; M-1 to M-75; M-1 to E-74;M-1 to T-73; M-1 to C-72; M-1 to N-71; M-1 to T-70; M-1 to F-69; M-1 toS-68; M-1 to E-67; M-1 to Y-66; M-1 to Y-65; M-1 to V-64; M-1 to I-63;M-1 to F-62; M-1 to E-61; M-1 to S-60; M-1 to L-59; M-1 to N-58; M-1 toC-57; M-1 to W-56; M-1 to K-55; M-1 to W-54; M-1 to V-53; M-1 to D-52;M-1 to V-51; M-1 to K-50; M-1 to G-49; M-1 to M-48; M-1 to M-47; M-1 toD-46; M-1 to A-45; M-1 to F-44; M-1 to A-43; M-1 to K-42; M-1 to G-41;M-1 to C-40; M-1 to L-39; M-1 to P-38; M-1 to L-37; M-1 to R-36; M-1 toE-35; M-1 to L-34; M-1 to M-33; M-1 to G-32; M-1 to T-31; M-1 to E-30;M-1 to N-29; M-1 to C-28; M-1 to G-27; M-1 to G-26; M-1 to A-25; M-1 toR-24; M-1 to P-23; M-1 to C-22; M-1 to G-21; M-1 to G-20; M-1 to C-19;M-1 to L-18; M-1 to L-17; M-1 to L-16; M-1 to L-15; M-1 to L-14; M-1 toP-13; M-1 to L-12; M-1 to L-11; M-1 to Q-10; M-1 to P-9; M-1 to R-8; M-1to R-7; of SEQ ID NO:31. Polynucleotides encoding these polypeptides arealso encompassed by the invention, as are antibodies that bind one ormore of these polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:31 whichhave been determined from the following related cDNA genes: HLHCH17RA(SEQ ID NO:76), HTOAT51R (SEQ ID NO:77), and/or HBNBO41R (SEQ ID NO:78).

The polypeptide of this gene has been determined to have a transmembranedomain at about amino acid position 122-138 of the amino acid sequencereferenced in Table XIV for this gene. Moreover, a cytoplasmic tailencompassing amino acids 139 to 149 of this protein has also beendetermined. Based upon these characteristics, it is believed that theprotein product of this gene shares structural features to type Iamembrane proteins.

Northern analysis indicates that a 1.4 kb transcript of this gene isprimarily expressed in small intestine tissue, and to a lesser extent incolon and prostate tissue.

Therefore, polynucleotides and polypeptides of the invention, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of diseases and conditions which include, but are not limitedto, gastrointestinal and neurodegenerative diseases and disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful in providing immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the centralnervous and gastrointestinal systems, expression of this gene atsignificantly higher or lower levels is routinely detected in certaintissues or cell types (e.g., brain, CNS, gastrointestinal, cancerous andwounded tissues) or bodily fluids (e.g., lymph, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution and homology to RAMP-3 suggest thatpolynucleotides, translation products and antibodies corresponding tothis gene are useful for the detection/treatment of neurodegenerativedisease states and behavioural disorders such as Alzheimer's Disease,Parkinson's Disease, Huntington's Disease, Tourette Syndrome,schizophrenia, mania, dementia, paranoia, obsessive compulsive disorder,panic disorder, learning disabilities, ALS, psychoses, autism, andaltered behaviors, including disorders in feeding, sleep patterns,balance, and perception. In addition, the gene or gene product may alsoplay a role in the treatment and/or detection of developmental disordersassociated with the developing embryo.

Alternatively, the tissue distribution in small intestine and colontissues indicates that polynucleotides, translation products andantibodies corresponding to this gene are useful for the diagnosisand/or treatment of disorders involving the small intestine. This mayinclude diseases associated with digestion and food absorption, as wellas hematopoietic disorders involving the Peyer's patches of the smallintestine, or other hematopoietic cells and tissues within the body.Similarly, expression of this gene product in colon tissue indicatesagain involvement in digestion, processing, and elimination of food, aswell as a potential role for this gene as a diagnostic marker orcausative agent in the development of colon cancer, and cancer ingeneral. Protein, as well as, antibodies directed against the proteinmay show utility as a tumor marker and/or immunotherapy targets for theabove listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:13 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1325 of SEQID NO:13, b is an integer of 15 to 1339, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:13, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 4

Translation products corresponding to this gene share sequence homologywith a proteoglycan from Gallus gallus, and this proteoglycan isbelieved to participate in the osteogenic processes of cartilageossification (See Genbank Accession No. gi|222847). Based on thesequence similarity, translation products corresponding to this gene areexpected to share biological activities with the Gallus gallusproteoglycan polypeptide.

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, or all four of theimmunogenic epitopes shown in SEQ ID NO: 32 as residues: Leu-22 toAsp-39, Asn-64 to Pro-76, Pro-98 to Thr-111, and/or Pro-291 to Glu-302.Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

FIGS. 10A-B shows the nucleotide (SEQ ID NO:14) and deduced amino acidsequence (SEQ ID NO:32) of the retinal specific protein. Predicted aminoacids from about 1 to about 21 constitute the predicted signal peptide(amino acid residues from about 1 to about 21 in SEQ ID NO:32) and arerepresented by the underlined amino acid regions.

FIG. 11 shows the regions of similarity between the amino acid sequencesof the retinal specific protein SEQ ID NO:32, and the Gallus gallusproteoglycan (SEQ ID NO:79).

FIG. 12 shows an analysis of the amino acid sequence of SEQ ID NO: 32.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the polypeptide having the aminoacid sequence shown in FIGS. 10A-B (SEQ ID NO:32), which was determinedby sequencing a cloned cDNA (HARAO44). The nucleotide sequence shown inFIGS. 10A-B (SEQ ID NO:14) was obtained by sequencing a cloned cDNA(HARAO44), which was deposited on Nov. 17, 1998 at the American TypeCulture Collection, and given Accession Number 203484. The depositedgene is inserted in the pSport plasmid (Life Technologies, Rockville,Md.) using the SalI/NotI restriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO:14 is intended DNA fragments atleast about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:14. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:14. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini.

Representative examples of polynucleotide fragments of the inventioninclude, for example, fragments that comprise, or alternatively, consistof, a sequence from about nucleotide 1 to about 50, from about 51 toabout 100, from about 101 to about 150, from about 151 to about 200,from about 201 to about 250, from about 251 to about 300, from about 301to about 350, from about 351 to about 400, from about 401 to about 450,from about 451 to about 500, and from about 501 to about 550, and fromabout 551 to about 600, from about 601 to about 650, from about 651 toabout 700, from about 701 to about 750, from about 751 to about 800,from about 801 to about 850, from about 851 to about 900, from about 901to about 950, from about 951 to about 1000, from about 1001 to about1050, from about 1051 to about 1100, from about 1101 to about 1150, fromabout 1151 to about 1200, from about 1201 to about 1250, from about 1251to about 1300, from about 1301 to about 1350, from about 1351 to about1389, and from about 187 to about 1119 of SEQ ID NO:14, or thecomplementary strand thereto, or the cDNA contained in the depositedgene. In this context “about”includes the particularly recited ranges,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. In additional embodiments, thepolynucleotides of the invention encode functional attributes of thecorresponding protein.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions. The data representing the structural or functionalattributes of the protein set forth in FIG. 12 and/or Table IV, asdescribed above, was generated using the various modules and algorithmsof the DNA*STAR set on default parameters. In a preferred embodiment,the data presented in columns VIII, IX, XIII, and XIV of Table IV can beused to determine regions of the protein which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX, XIII, and/or XIV bychoosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

Certain preferred regions in these regards are set out in FIG. 12, butmay, as shown in Table IV, be represented or identified by using tabularrepresentations of the data presented in FIG. 12. The DNA*STAR computeralgorithm used to generate FIG. 12 (set on the original defaultparameters) was used to present the data in FIG. 12 in a tabular format(See Table IV). The tabular format of the data in FIG. 12 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 12 and in Table IVinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS.10A-B. As set out in FIG. 12 and in Table IV, such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, etc.) may still beretained. For example, the ability of shortened muteins to induce and/orbind to antibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a mutein with a large numberof deleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixamino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence shown in FIGS. 10A-B, up to the proline residue at positionnumber 327 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-332 of FIGS. 10A-B, where n1 is aninteger from 2 to 327 corresponding to the position of the amino acidresidue in FIGS. 10A-B (which is identical to the sequence shown as SEQID NO:32). N-terminal deletions of the polypeptide of the inventionshown as SEQ ID NO:32 include polypeptides comprising the amino acidsequence of residues: R-2 to T-332; L-3 to T-332; L-4 to T-332; A-5 toT-332; F-6 to T-332; L-7 to T-332; S-8 to T-332; L-9 to T-332; L-10 toT-332; A-11 to T-332; L-12 to T-332; V-13 to T-332; L-14 to T-332; Q-15to T-332; E-16 to T-332; T-17 to T-332; G-18 to T-332; T-19 to T-332;A-20 to T-332; S-21 to T-332; L-22 to T-332; P-23 to T-332; R-24 toT-332; K-25 to T-332; E-26 to T-332; R-27 to T-332; K-28 to T-332; R-29to T-332; R-30 to T-332; E-31 to T-332; E-32 to T-332; Q-33 to T-332;M-34 to T-332; P-35 to T-332; R-36 to T-332; E-37 to T-332; G-38 toT-332; D-39 to T-332; S-40 to T-332; F-41 to T-332; E-42 to T-332; V-43to T-332; L-44 to T-332; P-45 to T-332; L-46 to T-332; R-47 to T-332;N-48 to T-332; D-49 to T-332; V-50 to T-332; L-51 to T-332; N-52 toT-332; P-53 to T-332; D-54 to T-332; N-55 to T-332; Y-56 to T-332; G-57to T-332; E-58 to T-332; V-59 to T-332; I-60 to T-332; D-61 to T-332;L-62 to T-332; S-63 to T-332; N-64 to T-332; Y-65 to T-332; E-66 toT-332; E-67 to T-332; L-68 to T-332; T-69 to T-332; D-70 to T-332; Y-71to T-332; G-72 to T-332; D-73 to T-332; Q-74 to T-332; L-75 to T-332;P-76 to T-332; E-77 to T-332; V-78 to T-332; K-79 to T-332; V-80 toT-332; T-81 to T-332; S-82 to T-332; L-83 to T-332; A-84 to T-332; P-85to T-332; A-86 to T-332; T-87 to T-332; S-88 to T-332; I-89 to T-332;S-90 to T-332; P-91 to T-332; A-92 to T-332; K-93 to T-332; S-94 toT-332; T-95 to T-332; T-96 to T-332; A-97 to T-332; P-98 to T-332; G-99to T-332; T-100 to T-332; P-101 to T-332; S-102 to T-332; S-103 toT-332; N-104 to T-332; P-105 to T-332; T-106 to T-332; M-107 to T-332;T-108 to T-332; R-109 to T-332; P-110 to T-332; T-111 to T-332; T-112 toT-332; A-113 to T-332; G-114 to T-332; L-115 to T-332; L-116 to T-332;L-117 to T-332; S-118 to T-332; S-119 to T-332; Q-120 to T-332; P-121 toT-332; N-122 to T-332; H-123 to T-332; G-124 to T-332; L-125 to T-332;P-126 to T-332; T-127 to T-332; C-128 to T-332; L-129 to T-332; V-130 toT-332; C-131 to T-332; V-132 to T-332; C-133 to T-332; L-134 to T-332;G-135 to T-332; S-136 to T-332; S-137 to T-332; V-138 to T-332; Y-139 toT-332; C-140 to T-332; D-141 to T-332; D-142 to T-332; I-143 to T-332;D-144 to T-332; L-145 to T-332; E-146 to T-332; D-147 to T-332; I-148 toT-332; P-149 to T-332; P-150 to T-332; L-151 to T-332; P-152 to T-332;R-153 to T-332; R-154 to T-332; T-155 to T-332; A-156 to T-332; Y-157 toT-332; L-158 to T-332; Y-159 to T-332; A-160 to T-332; R-161 to T-332;F-162 to T-332; N-163 to T-332; R-164 to T-332; I-165 to T-332; S-166 toT-332; R-167 to T-332; I-168 to T-332; R-169 to T-332; A-170 to T-332;E-171 to T-332; D-172 to T-332; F-173 to T-332; K-174 to T-332; G-175 toT-332; L-176 to T-332; T-177 to T-332; K-178 to T-332; L-179 to T-332;K-180 to T-332; R-181 to T-332; I-182 to T-332; D-183 to T-332; L-184 toT-332; S-185 to T-332; N-186 to T-332; N-187 to T-332; L-188 to T-332;I-189 to T-332; S-190 to T-332; S-191 to T-332; I-192 to T-332; D-193 toT-332; N-194 to T-332; D-195 to T-332; A-196 to T-332; F-197 to T-332;R-198 to T-332; L-199 to T-332; L-200 to T-332; H-201 to T-332; A-202 toT-332; L-203 to T-332; Q-204 to T-332; D-205 to T-332; L-206 to T-332;I-207 to T-332; L-208 to T-332; P-209 to T-332; E-210 to T-332; N-211 toT-332; Q-212 to T-332; L-213 to T-332; E-214 to T-332; A-215 to T-332;L-216 to T-332; P-217 to T-332; V-218 to T-332; L-219 to T-332; P-220 toT-332; S-221 to T-332; G-222 to T-332; I-223 to T-332; E-224 to T-332;F-225 to T-332; L-226 to T-332; D-227 to T-332; V-228 to T-332; R-229 toT-332; L-230 to T-332; N-231 to T-332; R-232 to T-332; L-233 to T-332;Q-234 to T-332; S-235 to T-332; S-236 to T-332; G-237 to T-332; I-238 toT-332; Q-239 to T-332; P-240 to T-332; A-241 to T-332; A-242 to T-332;F-243 to T-332; R-244 to T-332; A-245 to T-332; M-246 to T-332; E-247 toT-332; K-248 to T-332; L-249 to T-332; Q-250 to T-332; F-251 to T-332;L-252 to T-332; Y-253 to T-332; L-254 to T-332; S-255 to T-332; D-256 toT-332; N-257 to T-332; L-258 to T-332; L-259 to T-332; D-260 to T-332;S-261 to T-332; I-262 to T-332; P-263 to T-332; G-264 to T-332; P-265 toT-332; L-266 to T-332; P-267 to T-332; P-268 to T-332; S-269 to T-332;L-270 to T-332; R-271 to T-332; S-272 to T-332; V-273 to T-332; H-274 toT-332; L-275 to T-332; Q-276 to T-332; N-277 to T-332; N-278 to T-332;L-279 to T-332; I-280 to T-332; E-281 to T-332; T-282 to T-332; M-283 toT-332; Q-284 to T-332; R-285 to T-332; D-286 to T-332; V-287 to T-332;F-288 to T-332; C-289 to T-332; D-290 to T-332; P-291 to T-332; E-292 toT-332; E-293 to T-332; H-294 to T-332; K-295 to T-332; H-296 to T-332;T-297 to T-332; R-298 to T-332; R-299 to T-332; Q-300 to T-332; L-301 toT-332; E-302 to T-332; D-303 to T-332; I-304 to T-332; R-305 to T-332;L-306 to T-332; D-307 to T-332; G-308 to T-332; N-309 to T-332; P-310 toT-332; I-311 to T-332; N-312 to T-332; L-313 to T-332; S-314 to T-332;L-315 to T-332; F-316 to T-332; P-317 to T-332; S-318 to T-332; A-319 toT-332; Y-320 to T-332; F-321 to T-332; C-322 to T-332; L-323 to T-332;P-324 to T-332; R-325 to T-332; L-326 to T-332; P-327 to T-332; of SEQID NO:32. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities (e.g., ability to illicit mitogenicactivity, induce differentiation of normal or malignant cells, bind toEGF receptors, etc.)), may still be retained. For example the ability toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptide generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a mutein with alarge number of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the polypeptide shown in FIGS. 10A-B, up to the glutamineresidue at position number 7, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m1 of FIGS. 10A-B,where m1 is an integer from 7 to 331 corresponding to the position ofthe amino acid residue in FIGS. 10A-B. Moreover, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of the amino acid sequence of C-terminal deletions of thepolypeptide of the invention shown as SEQ ID NO:32 include polypeptidescomprising the amino acid sequence of residues: M-1 to F-331; M-1 toR-330; M-1 to G-329; M-1 to I-328; M-1 to P-327; M-1 to L-326; M-1 toR-325; M-1 to P-324; M-1 to L-323; M-1 to C-322; M-1 to F-321; M-1 toY-320; M-1 to A-319; M-1 to S-318; M-1 to P-317; M-1 to F-316; M-1 toL-315; M-1 to S-314; M-1 to L-313; M-1 to N-312; M-1 to I-311; M-1 toP-310; M-1 to N-309; M-1 to G-308; M-1 to D-307; M-1 to L-306; M-1 toR-305; M-1 to I-304; M-1 to D-303; M-1 to E-302; M-1 to L-301; M-1 toQ-300; M-1 to R-299; M-1 to R-298; M-1 to T-297; M-1 to H-296; M-1 toK-295; M-1 to H-294; M-1 to E-293; M-1 to E-292; M-1 to P-291; M-1 toD-290; M-1 to C-289; M-1 to F-288; M-1 to V-287; M-1 to D-286; M-1 toR-285; M-1 to Q-284; M-1 to M-283; M-1 to T-282; M-1 to E-281; M-1 toI-280; M-1 to L-279; M-1 to N-278; M-1 to N-277; M-1 to Q-276; M-1 toL-275; M-1 to H-274; M-1 to V-273; M-1 to S-272; M-1 to R-271; M-1 toL-270; M-1 to S-269; M-1 to P-268; M-1 to P-267; M-1 to L-266; M-1 toP-265; M-1 to G-264; M-1 to P-263; M-1 to I-262; M-1 to S-261; M-1 toD-260; M-1 to L-259; M-1 to L-258; M-1 to N-257; M-1 to D-256; M-1 toS-255; M-1 to L-254; M-1 to Y-253; M-1 to L-252; M-1 to F-251; M-1 toQ-250; M-1 to L-249; M-1 to K-248; M-1 to E-247; M-1 to M-246; M-1 toA-245; M-1 to R-244; M-1 to F-243; M-1 to A-242; M-1 to A-241; M-1 toP-240; M-1 to Q-239; M-1 to I-238; M-1 to G-237; M-1 to S-236; M-1 toS-235; M-1 to Q-234; M-1 to L-233; M-1 to R-232; M-1 to N-231; M-1 toL-230; M-1 to R-229; M-1 to V-228; M-1 to D-227; M-1 to L-226; M-1 toF-225; M-1 to E-224; M-1 to I-223; M-1 to G-222; M-1 to S-221; M-1 toP-220; M-1 to L-219; M-1 to V-218; M-1 to P-217; M-1 to L-216; M-1 toA-215; M-1 to E-214; M-1 to L-213; M-1 to Q-212; M-1 to N-211; M-1 toE-210; M-1 to P-209; M-1 to L-208; M-1 to I-207; M-1 to L-206; M-1 toD-205; M-1 to Q-204; M-1 to L-203; M-1 to A-202; M-1 to H-201; M-1 toL-200; M-1 to L-199; M-1 to R-198; M-1 to F-197; M-1 to A-196; M-1 toD-195; M-1 to N-194; M-1 to D-193; M-1 to I-192; M-1 to S-191; M-1 toS-190; M-1 to I-189; M-1 to L-188; M-1 to N-187; M-1 to N-186; M-1 toS-185; M-1 to L-184; M-1 to D-183; M-1 to I-182; M-1 to R-181; M-1 toK-180; M-1 to L-179; M-1 to K-178; M-1 to T-177; M-1 to L-176; M-1 toG-175; M-1 to K-174; M-1 to F-173; M-1 to D-172; M-1 to E-171; M-1 toA-170; M-1 to R-169; M-1 to I-168; M-1 to R-167; M-1 to S-166; M-1 toI-165; M-1 to R-164; M-1 to N-163; M-1 to F-162; M-1 to R-161; M-1 toA-160; M-1 to Y-159; M-1 to L-158; M-1 to Y-157; M-1 to A-156; M-1 toT-155; M-1 to R-154; M-1 to R-153; M-1 to P-152; M-1 to L-151; M-1 toP-150; M-1 to P-149; M-1 to I-148; M-1 to D-147; M-1 to E-146; M-1 toL-145; M-1 to D-144; M-1 to I-143; M-1 to D-142; M-1 to D-141; M-1 toC-140; M-1 to Y-139; M-1 to V-138; M-1 to S-137; M-1 to S-136; M-1 toG-135; M-1 to L-134; M-1 to C-133; M-1 to V-132; M-1 to C-131; M-1 toV-130; M-1 to L-129; M-1 to C-128; M-1 to T-127; M-1 to P-126; M-1 toL-125; M-1 to G-124; M-1 to H-123; M-1 to N-122; M-1 to P-121; M-1 toQ-120; M-1 to S-119; M-1 to S-118; M-1 to L-117; M-1 to L-116; M-1 toL-115; M-1 to G-114; M-1 to A-113; M-1 to T-112; M-1 to T-111; M-1 toP-110; M-1 to R-109; M-1 to T-108; M-1 to M-107; M-1 to T-106; M-1 toP-105; M-1 to N-104; M-1 to S-103; M-1 to S-102; M-1 to P-101; M-1 toT-100; M-1 to G-99; M-1 to P-98; M-1 to A-97; M-1 to T-96; M-1 to T-95;M-1 to S-94; M-1 to K-93; M-1 to A-92; M-1 to P-91; M-1 to S-90; M-1 toI-89; M-1 to S-88; M-1 to T-87; M-1 to A-86; M-1 to P-85; M-1 to A-84;M-1 to L-83; M-1 to S-82; M-1 to T-81; M-1 to V-80; M-1 to K-79; M-1 toV-78; M-1 to E-77; M-1 to P-76; M-1 to L-75; M-1 to Q-74; M-1 to D-73;M-1 to G-72; M-1 to Y-71; M-1 to D-70; M-1 to T-69; M-1 to L-68; M-1 toE-67; M-1 to E-66; M-1 to Y-65; M-1 to N-64; M-1 to S-63; M-1 to L-62;M-1 to D-61; M-1 to I-60; M-1 to V-59; M-1 to E-58; M-1 to G-57; M-1 toY-56; M-1 to N-55; M-1 to D-54; M-1 to P-53; M-1 to N-52; M-1 to L-51;M-1 to V-50; M-1 to D-49; M-1 to N-48; M-1 to R-47; M-1 to L-46; M-1 toP-45; M-1 to L-44; M-1 to V-43; M-1 to E-42; M-1 to F-41; M-1 to S-40;M-1 to D-39; M-1 to G-38; M-1 to E-37; M-1 to R-36; M-1 to P-35; M-1 toM-34; M-1 to Q-33; M-1 to E-32; M-1 to E-31; M-1 to R-30; M-1 to R-29;M-1 to K-28; M-1 to R-27; M-1 to E-26; M-1 to K-25; M-1 to R-24; M-1 toP-23; M-1 to L-22; M-1 to S-21; M-1 to A-20; M-1 to T-19; M-1 to G-18;M-1 to T-17; M-1 to E-16; M-1 to Q-15; M-1 to L-14; M-1 to V-13; M-1 toL-12; M-1 to A-11; M-1 to L-10; M-1 to L-9; M-1 to S-8; M-1 to L-7; ofSEQ ID NO:32. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:14 whichhave been determined from the following related cDNA genes: HARAY79R(SEQ ID NO:80), HARAO44R (SEQ ID NO:81), HARAJ74R (SEQ ID NO:82),HARAO66R (SEQ ID NO:83), HARAN19R (SEQ ID NO:84), and HARAT78R (SEQ IDNO:85).

Northern analysis indicates that this gene is expressed in adrenalcortex and adrenal medulla tissues. This gene is also expressed inretinal tissue.

Therefore, polynucleotides and polypeptides of the invention, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of diseases and conditions which include, but are not limitedto, retinal disorders. Similarly, polypeptides and antibodies directedto these polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theretina, expression of this gene at significantly higher or lower levelsis routinely detected in certain tissues or cell types (e.g., retinal,cancerous and wounded tissues) or bodily fluids (e.g., lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution in retinal tissue, and the homology to a Gallusgallus proteoglycan involved in the ossification process indicates thatpolynucleotides, translation products and antibodies corresponding tothis gene are useful for the treatment of disorders of the retina whichinvolve the adhesion of tissues, or the binding of certain proteins tothe cell surface. Polynucleotides, translation products and antibodiescorresponding to this gene are useful for the treatment of retinaldisorders such as retinal detachment in individuals suffering frommyopia, or in the treatment of macular degeneration. Furthermore, thisgene may serve as a tumor marker for retinoblastomas, or related tumors.More generally, the tissue distribution in retinal tissue indicates thatpolynucleotides, translation products and antibodies corresponding tothis gene are useful for the diagnosis, detection and/or treatment ofeye disorders including blindness, color blindness, impaired vision,short and long sightedness, retinitis pigmentosa, retinitis proliferans,and retinoblastoma, retinochoroiditis, retinopathy and retinoschisis.Based upon the tissue distribution of this protein, antagonists directedagainst this protein are useful in blocking the activity of thisprotein. Accordingly, preferred are antibodies which specifically bind aportion of the translation product of this gene.

Also provided is a kit for detecting tumors in which expression of thisprotein occurs. Such a kit comprises in one embodiment an antibodyspecific for the translation product of this gene bound to a solidsupport. Also provided is a method of detecting these tumors in anindividual which comprises a step of contacting an antibody specific forthe translation product of this gene to a bodily fluid from theindividual, preferably serum, and ascertaining whether antibody binds toan antigen found in the bodily fluid. Preferably the antibody is boundto a solid support and the bodily fluid is serum. The above embodiments,as well as other treatments and diagnostic tests (kits and methods), aremore particularly described elsewhere herein. Furthermore, the proteinmay also be used to determine biological activity, to raise antibodies,as tissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:14 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1375 of SEQID NO:14, b is an integer of 15 to 1389, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:14, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 5

Translation products corresponding to the CD33-like gene (SEQ ID NOs: 33and/or 149) share sequence homology with the CD33 protein (See GenbankAccession BAA24983). The expression pattern of CD33 within thehematopoietic system indicates a potential role in the regulation ofmyeloid cell differentiation. However, this expression is absent fromhematopoietic stem cells.

CD33 is expressed in clonogenic leukemia cells in about 90% of patientssuffering from acute myeloid leukemia (AML). While about 60-70% ofadults suffering from AML experience complete remission due tochemotherapy application, most of these patients will ultimately die ofrelapsed leukemia It is believed that, like CD33, the CD33-like proteinof the present invention is also expressed by clonogenic leukemia cellsfrom the vast majority of patients with AML. Thus, there is a clear needto identify and isolate nucleic acid molecules encoding additionalpolypeptides having CD33-like protein activity. It is believed thatcancerous tissue contains significantly greater amounts of CD33-likeprotein gene copy number and expresses significantly enhanced levels ofCD33-like protein and mRNA encoding the CD33-like protein when comparedto a “standard” mammal, i.e.—a mammal of the same species not having thecancer or inflammatory disease. Thus, enhanced levels of the CD33-likeprotein will be detected in certain bodily fluids (e.g., serum, plasma,urine, synovial fluid and spinal fluid) from mammals when compared tosera from mammals of the same species not having the cancer orinflammatory disease.

Translation products corresponding to the CD33-like and CD33-likeSV (asplice variant of CD33-like) (SEQ ID NOs: 33 and/or 149) share sequencehomology with a number of CD33-related Siglecs, namely Siglecs-3, -5(See Genbank Accession AAD50978), -6 (See Genbank Accession NP001236),-7 (See Genbank Accession AAF12759), -8 (See Genbank AccessionAAF27622), and -9 (See Genbank Accession AAF87223). Siglecs are sialicacid binding proteins, and are members of the Ig superfamily which areexpressed at the cellular surface. Each Siglec member exhibits distinctsialic acid binding properties, and has a characteristic tissueexpression profile. Based upon the homology, it is thought thatCD33-like and CD33-likeSV will share at least some of the biologicalactivities of other Siglecs (e.g., sialic acid binding). CD33-likeSV isthought to exist as a monomer in the plasma membrane (See FIG. 43 and/orExample 54). CD33-likeSV was mapped by in situ hybridization to the longarm of chromosome 19, in the 19q13.3 band, which is closely linked toother CD33-related Siglecs.

Two related cDNAs, HDPIB36 and HEOMH10, have been isolated. These cDNAsappear to encode splice variants of the same gene. Preferredpolynucleotides comprise, or alternatively consist of, a polynucleotidesequence selected from the following sequences:CGACCCACGCGTCCGCCGCCTTCGGCTTCCCCTTCTGC

CAAGAGCCCTGAGCCACTCACAGCACGACCAGAGA (SEQ ID NO: 86),

GTATGGAATGGGGTGGGAACCCCTGCCTCTCACACTGGGGAGGGACCCTGGGGACAGCCTATGGGCTGAGCAGAGAGGGCTCTCAGGGACCCCTGCAGCACAAGAATCTCCCACCACGGTCTCTGTCCCAGCCCTGACTCAGAAGCCTGATGTCTACATCCCCGAGACCCTGGAGCCCGGGCAGCCGGTGACGGTCATCTGTGTGTTTAACTGGGCCTTTGAGGAATGTCCACCCCCTTCTTTCTCCTGGACGGGGGCTGCCCTCTCCTCCCAAGGAACCAAACCAACGACCTCCCACTTCTCAG (SEQ ID NO: 87),

ATCCTCCAGAGAACCTGAGAGTGATGGTTTCCCAAGCAAACAGGACAGGTAGGAAAGGGGACAGAGGAGCCAAGGCCTCTCAGTGCCGAATTGGGGGCCCAGGAGTCTGGAGGGTCCCCACGCAGGAGGGTCCCTGAGCCCTGAGCTGCTCATCGATTCTGCCTCTTCCTTCCCT (SEQ ID NO: 88),

GTGAGTGGGGGAAAGGGGACACCTGGGTCCCAGGAAGGGGACCCTGCTGAGTCCTGTCCTCCCTCCCCTCAG (SEQ ID NO: 89),

CTGGCCCCCTGGCTCAGAAGCGGAATCAGAAAGCCACACCAAACAGTCCTCGGACCCCTCTTCCACCAGGTGCTCCCTCCCCAGAATCAAAGAAGAACCAGAAAAAGCAGTATCAGTTGCCCAGTTTCCCAGAACCCAAATCATCCACTCAAGCCCCAGAATCCCAGGAGAGCCAAGAGGAGCTCCATTATGCCACGCTCAACTTCCCAGGCGTCAGACCCAGGCCTGAGGCCCGGATGCCCAAGGGCACCCAGGCGGATTATGCAGAAGTCAAGTTCCAATGAGGGTCTCTTAGGCTTTAGGACTGGGACTTCGGCTAGGGAGGAAGGTAGAGTAAGAGGTTGAAGATAACAGAGTGCAAAGTTTCCTTCTCTCCCTCTCTCTCTCTCTTTCTCTCTCTCTCTCTCTTTCTCTCTCTTTT (SEQ ID NO: 90),and/or

AAAAAAACATCTGGCCAGGGCACAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGTTGAGGTGGGCAGATCGCCTGAGGTCGGGAGTTCGAGACCAGCCTGGCCAACTTGGTGAAACCCCGTCTCTACTAAAAATACAAAAATTAGCTGGGCATGGTGGCAGGCGCCTGTAATCCTACTACTTGGGAAGCTGAGGCAGGAGAATCACTTGAACCTGGGAGACGGAGGTTGCAGTGAGCCAAGATCACACCATTGCACGCCAGCTTGGGCAACAAAGCGAGACTCCATCTCAAAAAAAAAATCCTCCAAATGGGTTGGGTGTCTGTAATCCCAGCACTTTGGGAGGCTAAGGTGGGTGGATTGCTTGAGCCCAGGAGTTCGAGACCAGCCTGGGCAACATGGTGAAACCCCATCTCTACAAAAAATACAAAACATAGCTGGGCTTGGTGGTGTGTGCCTGTAGTCCCAGCTGTCAGACATTTAAACCAGAGCAACTCCCATCTGGAATGGGAGCTGAATAAAATGAGGCTGAGACCTACTGGGCTGCCATTCTCAGACAGTGGAGGCCATTCTAAGTCACAGGATGAGACAGGAGGTCCGTACAAGATACAGGTCATAAAGACTTTGCTGATAAAACAGATTGCAGTAAAGAAGCCAACCAAATCCCACCAAAACCAAGTTGGCCACGAGAGTGACCTCTGGTCGTCCTCACTGCTACACTCCTGACAGCACCATGACAGTTTACAAATGCCATGGCAACATCAGGAAGTTACCCGATATGTCCCAAAAGGGGGAGGAATGAATAATCCACCCCTTGTTTAGCAAATAAGCAAGAAATAACCATAAAAGTGGGCAACCAGCAGCTCTAGGCGCTGCTCTTGTCTATGGAGTAGCCATTCTTTTGTTCCTI TACTTTCTTAATAAACTTGCTTTCACCTT (SEQ ID NO:91). Alsopreferred are the polypeptides encoded by these polynucleotides.

Preferred CD33-like polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, or all twenty of the predictedimmunogenic epitopes shown in SEQ ID NO: 33 as residues: Pro-46 toGly-52, Asn-76 to Val-82, Ser-85 to Phe-90, Gly-94 to Asn-100, Gln-111to Tyr-116, Pro-146 to Leu-155, Ser-188 to Asn-202, Ser-240 to Arg-246,Gly-258 to Tyr-263, Ala-267 to Arg-276, Ser-326 to Arg-331, Ser-333 toGln-339, Pro-343 to Asp-348, Glu-426 to Asp-432, Pro-517 to His-533,Ala-550 to Pro-565, Gly-569 to Gln-582, Pro-589 to Glu-606, Gly-616 toAla-623, and/or Met-625 to Ala-631. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g. fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

Preferred CD33-likeSV polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, or alltwenty-two of the predicted immunogenic epitopes shown in SEQ ID NO: 149as residues: Pro-46 to Gly-52, Asn-76 to Val-82, Ser-85 to Phe-90,Gly-94 to Asn-100, Gln-111 to Tyr-116, Cys-173 to Ser-179, Gln-188 toSer-195, Pro-204 to Leu-213, Ser-246 to Asn-260, Ser-298 to Arg-304,Gly-316 to Tyr-321, Ala-325 to Arg-334, Ser-384 to Arg-389, Ser-391 toGln-397, Pro-401 to Asp-406, Glu-484 to Asp-490, Pro-575 to His-591,Ala-608 to Pro-623, Gly-627 to Gln-640, Pro-647 to Glu-664, Gly-674 toAla-681, and Met-683 to Ala-689. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g. fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

FIGS. 13A-C show the nucleotide (SEQ ID NO:15) and deduced amino acidsequence (SEQ ID NO:33) of the CD33-like protein. Predicted amino acidsfrom about 1 to about 16 constitute the predicted signal peptide (aminoacid residues from about 1 to about 16 in SEQ ID NO:33) and arerepresented by the underlined amino acid regions; and amino acids fromabout 496 to about 512 constitute the predicted transmembrane domain(amino acid residues from about 496 to about 512 in SEQ ID NO:33) andare represented by the double-underlined amino acid regions.

FIG. 14 shows the regions of similarity between the amino acid sequencesof the CD33-like protein SEQ ID NO:33, and the CD33L1 protein (SeeGenbank Accession A30521) (SEQ ID NO: 92).

FIG. 15 shows an analysis of the amino acid sequence of CD33-likeprotein (SEQ ID NO:33). Alpha, beta, turn and coil regions;hydrophilicity and hydrophobicity; amphipathic regions; flexibleregions; antigenic index and surface probability are shown.

FIG. 37 shows the predicted protein sequence of CD33-likeSV protein(identified in FIG. 37 as “Siglec-10”) and alignment withclosely-related Siglecs. Alignment was performed with the ClustalWmultiple sequence alignment program. Residues that are identical in morethan half the proteins are boxed in black, similar residues in grey.Asterisks indicate positions of the cysteine residues characteristic ofSiglecs. Filled circles overlay residues important for sialic acidbinding. Vertical lines indicate positions of intron-exon boundaries, asdeduced from the sequence of the gene encoding CD33-likeSV. Positions ofthe domain boundaries, transmembrane region, cytoplasmic tail (encodedby two exons) and the tyrosine based motifs are indicated. Genbankaccession numbers are as follows: CD33/Siglec-3, Siglec-5: AAD50978;Siglec-6, NP001236; Siglec-7 AAF12759; Siglec-8, AAF27622; and Siglec-9,AAF87223.

FIG. 38 provides a phylogenetic analysis of CD33-related Siglecs andCD33-likeSV protein (identified in FIG. 38 as “Siglec-10”). The leaderpeptide, domain 1, and domain 2, or the transmembrane and cytoplasmictails were aligned using the Clustal W multiple sequence alignmentprogram and analyzed for phylogenetic relationship using the PHYLIP 3.6.Unrooted phylograms were constructed using the neighbor joining method.

FIG. 39 provides a sequential deletion model for evolution ofCD33-related Siglecs. Based on the phylogenetic analysis and sequencecomparisons, this model predicts that MAG and CD33-likeSV (identified inFIG. 39 as “Siglec-10”) are both derived from a common 4-domainprogenitor. CD33-likeSV then gave rise to Siglec-5 involving a deletionof the exons encoding domain 3 and its associated linker. Siglec-5 thengave rise to a three domain siglec by deletion of domain 4. The other3-domain Siglecs then arose through gene duplication. CD33 may have beenderived in a single deletion event either from Siglec-5 or one of thethree domain Siglecs.

FIGS. 40A & B provides the localization and expression of theCD33-likeSV gene.

Human lymphocyte metaphase spreads were hybridized with a 3 kbbiotinylated insert from HEONM10 followed by fluorescein-avidin and thechromosomes counterstained with propidium iodide. The digital image isreversed to illustrate the hybridization signals (arrows) on the longarm of chromosome 19. The position of CD33-likeSV on chromosome 19 bandq13.3 is also shown schematically.

Northern blot analysis of CD33-likeSV mRNA in human tissues. Each laneof the Multiple Tissue Northern (MTN) Blot (Clontech) containsapproximately 2 gg poly A+ RNA from the tissue indicated and isnormalized for levels of β-actin mRNA. A major form of CD33-likeSV mRNAis seen at around 3.0 kb in most tissues.

FIG. 41 disclosed the binding of CD33-likeSV (identified in FIG. 41 as“Siglec-10”) expressed on COS cells to polyacrylamide conjugates. CR1was included as a negative control to measure non-specific binding.Three days after transient transfection, COS cells expressing theindicated proteins were incubated with biotinylated polyacrylamide (PAA)glycoconjugates linked either to 3′ sialyllactose (2,3-PAA) or 6′sialyllactose (2,6-PAA) or lactose (Lac-PAA) at 20 μg/ml or with bufferalone. Unbound conjugate was washed off and binding detected with¹²⁵I-streptavidin. Data show means standard deviations of quadruplicatesand are representative of 3 experiments performed.

FIGS. 42A, B & C show the expression of CD33-likeSV (identified in FIG.42 as “Siglec-10”) on human peripheral blood leukocyte subsets.

FACS histograms showing expression of CD33-likeSV on granulocytes,monocytes and lymphocytes, gated in each case according to theircharacteristic side and forward scatter properties. Thick lines showstaining with affinity purified mouse anti-CD33-likeSV polyclonalantibody. Thin lines show staining in the presence of mouse IgG used asa negative control. CD33-likeSV is expressed on a minor subset ofgranulocytes, most monocytes and a subset of lymphocytes

Double labeling of granulocytes with anti-CD 16 (neutrophils) andanti-CD33-likeSV mAb compared with anti-Siglec-8 mAb. Compared to theisotype matched control mAb, CD33-likeSV shows clear labeling of theeosinophils and some of the neutrophils are also weakly stained. Valuesrepresent the percentages of total granulocytes analyzed.

Double labeling of the lymphocyte fraction with antibodies to CD 19 (Bcells), CD3 (pan T cell), CD4 and CD8 (T cell subsets) and CD56 (NKcells). CD33-likeSV is expressed by most CD 19+ B cells and a smallsubset of CD16+ cells that do not express the CD56 natural killer cellmarker. Values represent the percentages of the total lymphocytesanalyzed. Similar results were obtained using the mouse anti-CD33-likeSVmAb, 5G6.

FIG. 43 provides a molecular characterization of CD33-likeSV (identifiedin FIG. 43 as “S10”). Stably-transfected CHO cells expressingCD33-likeSV, wild-type CHO cells, or Daudi cells were surfacebiotinylated, lysed and immunoprecipitations performed with mouseanti-CD33-likeSV polyclonal antibody. Precipitates were run either underreducing or non-reducing conditions on 4-12% gradient SDS polyacrylamidegels, transferred to nitrocellulose and probed with streptavidin-horseradish peroxidase. CD33-likeSV migrates as a single monomeric species ataround 120 kDa in CHO cells and around 100 kDa in Daudi cells.

FIG. 44 shows an analysis of the amino acid sequence of CD33-likeSVprotein (SEQ ID NO: 149). Alpha, beta, turn and coil regions;hydrophilicity and hydrophobicity; amphipathic regions; flexibleregions; antigenic index and surface probability are shown.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the polypeptide having the aminoacid sequence shown in FIGS. 13A-C (SEQ ID NO:33), which was determinedby sequencing a cloned cDNA (HDPCL05). The nucleotide sequence shown inFIGS. 13A-C (SEQ ID NO:15) was obtained by sequencing a cloned cDNA(HDPCL05), which was deposited on Nov. 17, 1998 at the American TypeCulture Collection, and given Accession Number 203484. The depositedgene is inserted in the pSport plasmid (Life Technologies, Rockville,Md.) using the SalI/NotI restriction endonuclease cleavage sites.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the polypeptide having the aminoacid sequence shown in FIG. 37 (SEQ ID NO:149).

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA, thenucleotide sequence shown in SEQ ID NO:15, or the nucleotide sequenceshown in SEQ ID NO: 148, is intended DNA fragments at least about 15 nt,and more preferably at least about 20 nt, still more preferably at leastabout 30 nt, and even more preferably, at least about 40 nt in lengthwhich are useful as diagnostic probes and primers as discussed herein.Of course, larger fragments 50-1500 nt in length are also usefulaccording to the present invention, as are fragments corresponding tomost, if not all, of the nucleotide sequence of the deposited cDNA, asshown in SEQ ID NO:15, or as shown in SEQ ID NO: 148. By a fragment atleast 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from the nucleotide sequence of thedeposited cDNA or the nucleotide sequence as shown in SEQ ID NO:15. Inthis context “about” includes the particularly recited size, larger orsmaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus orat both termini. Representative examples of polynucleotide fragments ofthe invention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, from about 501 toabout 550, from about 551 to about 600, from about 601 to about 650,from about 651 to about 700, from about 701 to about 750, from about 751to about 800, from about 801 to about 850, from about 851 to about 900,from about 901 to about 950, from about 951 to about 1000, from about1001 to about 1050, from about 1051 to about 1100, from about 1101 toabout 1150, from about 1151 to about 1200, from about 1201 to about1250, from about 1251 to about 1300, from about 1301 to about 1350, fromabout 1351 to about 1400, from about 1401 to about 1450, from about 1451to about 1500, from about 1501 to about 1550, from about 1551 to about1600, from about 1601 to about 1650, from about 1651 to about 1700, fromabout 1701 to about 1750, from about 1751 to about 1800, from about 1801to about 1850, from about 1851 to about 1900, from about 1901 to about1950, from about 1951 to about 2000, from about 2001 to about 2050, fromabout 2051 to about 2100, from about 2101 to about 2150, from about 2151to about 2200, from about 2201 to about 2250, from about 2251 to about2295, from about 307 to about 1977, and from about 106 to about 1977, ofSEQ ID NO:15, of SEQ ID NO: 148, or the complementary strand thereto, orthe cDNA contained in the deposited gene. In this context “about”includes the particularly recited ranges, larger or smaller by several(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.In additional embodiments, the polynucleotides of the invention encodefunctional attributes of the corresponding protein.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions. The data representing the structural or functionalattributes of the protein set forth in FIG. 15, FIG. 44, and/or Tables Vor XIII, as described above, was generated using the various modules andalgorithms of the DNA*STAR set on default parameters. In a preferredembodiment, the data presented in columns VII, IX, XIII, and XIV ofTables V and XIII can be used to determine regions of the protein whichexhibit a high degree of potential for antigenicity. Regions of highantigenicity are determined from the data presented in columns VIII, IX,XIII, and/or XIV by choosing values which represent regions of thepolypeptide which are likely to be exposed on the surface of thepolypeptide in an environment in which antigen recognition may occur inthe process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIGS. 15 and44, but may, as shown in Tables V and XIII, be represented or identifiedby using tabular representations of the data presented in FIGS. 15 and44. The DNA*STAR computer algorithm used to generate FIGS. 15 and 44,(set on the original default parameters) was used to present the data inFIGS. 15 and 44 in a tabular format (See Tables V and XIII). The tabularformat of the data in FIGS. 15 and 44 is used to easily determinespecific boundaries of a preferred region. The above-mentioned preferredregions set out in FIGS. 15 and 44 and in Tables V and XIII include, butare not limited to, regions of the aforementioned types identified byanalysis of the amino acid sequences set out in FIGS. 13A-C and FIG. 37.As set out in FIGS. 15 and 44, and in Tables V and XIII, such preferredregions include Garnier-Robson alpha-regions, beta-regions,turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions,and turn-regions, Kyte-Doolittle hydrophilic regions and Hopp-Woodshydrophobic regions, Eisenberg alpha- and beta-amphipathic regions,Karplus-Schulz flexible regions, Jameson-Wolf regions of high antigenicindex and Emini surface-forming regions. Even if deletion of one or moreamino acids from the N-terminus of a protein results in modification ofloss of one or more biological functions of the protein, otherfunctional activities (e.g., biological activities, ability tomultimerize, etc.) may still be retained. For example, the ability ofshortened muteins to induce and/or bind to antibodies which recognizethe complete or mature forms of the polypeptides generally will beretained when less than the majority of the residues of the complete ormature polypeptide are removed from the N-terminus. Whether a particularpolypeptide lacking N-terminal residues of a complete polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that a mutein with a large number of deleted N-terminal aminoacid residues may retain some biological or immunogenic activities. Infact, peptides composed of as few as six amino acid residues may oftenevoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence shown in FIGS. 13A-C, up to the alanine residue at positionnumber 634 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-639 of FIGS. 13A-C, where n1 is aninteger from 2 to 634 corresponding to the position of the amino acidresidue in FIGS. 13A-C (which is identical to the sequence shown as SEQID NO:33). N-terminal deletions of the polypeptide of the inventionshown as SEQ ID NO:33 include polypeptides comprising, or alternativelyconsisting of, an amino acid sequence selected from residues: L-2 toQ-639; L-3 to Q-639; P-4 to Q-639; L-5 to Q-639; L-6 to Q-639; L-7 toQ-639; S-8 to Q-639; S-9 to Q-639; L-10 to Q-639; L-11 to Q-639; G-12 toQ-639; G-13 to Q-639; S-14 to Q-639; Q-15 to Q-639; A-16 to Q-639; M-17to Q-639; D-18 to Q-639; G-19 to Q-639; R-20 to Q-639; F-21 to Q-639;W-22 to Q-639; I-23 to Q-639; R-24 to Q-639; V-25 to Q-639; Q-26 toQ-639; E-27 to Q-639; S-28 to Q-639; V-29 to Q-639; M-30 to Q-639; V-31to Q-639; P-32 to Q-639; E-33 to Q-639; A-34 to Q-639; C-35 to Q-639;D-36 to Q-639; I-37 to Q-639; S-38 to Q-639; V-39 to Q-639; P-40 toQ-639; C41 to Q-639; S-42 to Q-639; F-43 to Q-639; S-44 to Q-639; Y-45to Q-639; P-46 to Q-639; R-47 to Q-639; Q-48 to Q-639; D-49 to Q-639;W-50 to Q-639; T-51 to Q-639; G-52 to Q-639; S-53 to Q-639; T-54 toQ-639; P-55 to Q-639; A-56 to Q-639; Y-57 to Q-639; G-58 to Q-639; Y-59to Q-639; W-60 to Q-639; F-61 to Q-639; K-62 to Q-639; A-63 to Q-639;V-64 to Q-639; T-65 to Q-639; E-66 to Q-639; T-67 to Q-639; T-68 toQ-639; K-69 to Q-639; G-70 to Q-639; A-71 to Q-639; P-72 to Q-639; V-73to Q-639; A-74 to Q-639; T-75 to Q-639; N-76 to Q-639; H-77 to Q-639;Q-78 to Q-639; S-79 to Q-639; R-80 to Q-639; E-81 to Q-639; V-82 toQ-639; E-83 to Q-639; M-84 to Q-639; S-85 to Q-639; T-86 to Q-639; R-87to Q-639; G-88 to Q-639; R-89 to Q-639; F-90 to Q-639; Q-91 to Q-639;L-92 to Q-639; T-93 to Q-639; G-94 to Q-639; D-95 to Q-639; P-96 toQ-639; A-97 to Q-639; K-98 to Q-639; G-99 to Q-639; N-100 to Q-639;C-101 to Q-639; S-102 to Q-639; L-103 to Q-639; V-104 to Q-639; I-105 toQ-639; R-106 to Q-639; D-107 to Q-639; A-108 to Q-639; Q-109to Q-639;M-110 to Q-639; Q-111 to Q-639; D-112 to Q-639; E-113 to Q-639; S-114 toQ-639; Q-115 to Q-639; Y-116 to Q-639; F-117 to Q-639; F-118 to Q-639;R-119 to Q-639; V-120 to Q-639; E-121 to Q-639; R-122 to Q-639; G-123 toQ-639; S-124 to Q-639; Y-125 to Q-639; V-126 to Q-639; R-127 to Q-639;Y-128 to Q-639; N-129 to Q-639; F-130 to Q-639; M-131 to Q-639; N-132 toQ-639; D-133 to Q-639; G-134 to Q-639; F-135 to Q-639; F-136 to Q-639;L-137 to Q-639; K-138 to Q-639; V-139to Q-639; T-140 to Q-639; V-141 toQ-639; L-142 to Q-639; S-143 to Q-639; F-144 to Q-639; T-145 to Q-639;P-146 to Q-639; R-147to Q-639; P-148 to Q-639; Q-149 to Q-639; D-150 toQ-639; H-151 to Q-639; N-152 to Q-639; T-153 to Q-639; D-154 to Q-639;L-155 to Q-639; T-156 to Q-639; C-157 to Q-639; H-158 to Q-639; V-159 toQ-639; D-160 to Q-639; F-161 to Q-639; S-162to Q-639; R-163 to Q-639;K-164 to Q-639; G-165 to Q-639; V-166 to Q-639; S-167 to Q-639; A-168 toQ-639; Q-169 to Q-639; R-170 to Q-639; T-171 to Q-639; V-172 to Q-639;R-173 to Q-639; L-174 to Q-639; R-175 to Q-639; V-176 to Q-639; A-177 toQ-639; Y-178 to Q-639; A-179 to Q-639; P-180 to Q-639; R-181 to Q-639;D-182 to Q-639; L-183 to Q-639; V-184to Q-639; I-185 to Q-639; S-186 toQ-639; I-187 to Q-639; S-188 to Q-639; R-189 to Q-639; D-190 to Q-639;N-191 to Q-639; T-192 to Q-639; P-193 to Q-639; A-194 to Q-639; L-195 toQ-639; E-196 to Q-639; P-1 97 to Q-639; Q-198 to Q-639; P-199 to Q-639;Q-200 to Q-639; G-201 to Q-639; N-202 to Q-639; V-203 to Q-639; P-204 toQ-639; Y-205 to Q-639; L-206 to Q-639; E-207 to Q-639; A-208 to Q-639;Q-209 to Q-639; K-210 to Q-639; G-211 to Q-639; Q-212 to Q-639; F-213 toQ-639; L-214 to Q-639; R-215 to Q-639; L-216 to Q-639; L-217 to Q-639;C-218 to Q-639; A-219 to Q-639; A-220 to Q-639; D-221 to Q-639; S-222 toQ-639; Q-223 to Q-639; P-224 to Q-639; P-225 to Q-639; A-226 to Q-639;T-227 to Q-639; L-228 to Q-639; S-229 to Q-639; W-230 to Q-639; V-231 toQ-639; L-232 to Q-639; Q-233 to Q-639; N-234 to Q-639; R-235 to Q-639;V-236 to Q-639; L-237 to Q-639; S-238 to Q-639; S-239 to Q-639; S-240 toQ-639; H-241 to Q-639; P-242to Q-639; W-243 to Q-639; G-244 to Q-639;P-245 to Q-639; R-246 to Q-639; P-247 to Q-639; L-248 to Q-639; G-249 toQ-639; L-250 to Q-639; E-251 to Q-639; L-252 to Q-639; P-253 to Q-639;G-254 to Q-639; V-255 to Q-639; K-256 to Q-639; A-257 to Q-639; G-258 toQ-639; D-259 to Q-639; S-260 to Q-639; G-261 to Q-639; R-262 to Q-639;Y-263 to Q-639; T-264 to Q-639; C-265 to Q-639; R-266 to Q-639; A-267 toQ-639; E-268 to Q-639; N-269 to Q-639; R-270 to Q-639; L-271 to Q-639;G-272 to Q-639; S-273 to Q-639; Q-274 to Q-639; Q-275 to Q-639; R-276 toQ-639; A-277 to Q-639; L-278 to Q-639; D-279 to Q-639; L-280 to Q-639;S-281 to Q-639; V-282 to Q-639; Q-283 to Q-639; Y-284 to Q-639; P-285 toQ-639; P-286 to Q-639; E-287to Q-639; N-288 to Q-639; L-289 to Q-639;R-290 to Q-639; V-291 to Q-639; M-292 to Q-639; V-293 to Q-639; S-294 toQ-639; Q-295 to Q-639; A-296 to Q-639; N-297 to Q-639; R-298 to Q-639;T-299 to Q-639; V-300 to Q-639; L-301 to Q-639; E-302 to Q-639; N-303 toQ-639; L-304 to Q-639; G-305 to Q-639; N-306 to Q-639; G-307 to Q-639;T-308 to Q-639; S-309 to Q-639; L-310 to Q-639; P-311 to Q-639; V-312 toQ-639; L-313 to Q-639; E-314 to Q-639; G-315 to Q-639; Q-316 to Q-639;S-317 to Q-639; L-318 to Q-639; C-319 to Q-639; L-320 to Q-639; V-321 toQ-639; C-322 to Q-639; V-323 to Q-639; T-324 to Q-639; H-325 to Q-639;S-326 to Q-639; S-327 to Q-639; P-328 to Q-639; P-329 to Q-639; A-330 toQ-639; R-331 to Q-639; L-332 to Q-639; S-333 to Q-639; W-334 to Q-639;T-335 to Q-639; Q-336 to Q-639; R-337 to Q-639; G-338 to Q-639; Q-339 toQ-639; V-340 to Q-639; L-341 to Q-639; S-342 to Q-639; P-343 to Q-639;S-344 to Q-639; Q-345 to Q-639; P-346 to Q-639; S-347 to Q-639; D-348 toQ-639; P-349 to Q-639; G-350 to Q-639; V-351 to Q-639; L-352 to Q-639;E-353 to Q-639; L-354 to Q-639; P-355 to Q-639; R-356 to Q-639; V-357 toQ-639; Q-358 to Q-639; V-359 to Q-639; E-360 to Q-639; H-361 to Q-639;E-362 to Q-639; G-363 to Q-639; E-364 to Q-639; F-365 to Q-639; T-366 toQ-639; C-367 to Q-639; H-368 to Q-639; A-369 to Q-639; R-370 to Q-639;H-371 to Q-639; P-372 to Q-639; L-373 to Q-639; G-374 to Q-639; S-375 toQ-639; Q-376 to Q-639; H-377 to Q-639; V-378 to Q-639; S-379 to Q-639;L-380 to Q-639; S-381 to Q-639; L-382 to Q-639; S-383 to Q-639; V-384 toQ-639; H-385 to Q-639; Y-386 to Q-639; S-387 to Q-639; P-388 to Q-639;K-389 to Q-639; L-390 to Q-639; L-391 to Q-639; G-392 to Q-639; P-393 toQ-639; S-394 to Q-639; C-395 to Q-639; S-396 to Q-639; W-397 to Q-639;E-398 to Q-639; A-399 to Q-639; E-400 to Q-639; G-401 to Q-639; L-402 toQ-639; H-403 to Q-639; C-404 to Q-639; S-405 to Q-639; C-406 to Q-639;S-407 to Q-639; S-408 to Q-639; Q-409 to Q-639; A-410 to Q-639; S-411 toQ-639; P-412 to Q-639; A-413 to Q-639; P-414 to Q-639; S-415 to Q-639;L-416 to Q-639; R-417 to Q-639; W-418 to Q-639; W-419 to Q-639; L-420 toQ-639; G-421 to Q-639; E-422 to Q-639; E-423 to Q-639; L-424to Q-639;L-425 to Q-639; E-426 to Q-639; G-427 to Q-639; N-428 to Q-639; S-429 toQ-639; S-430 to Q-639; Q-431 to Q-639; D-432 to Q-639; S-433 to Q-639;F-434 to Q-639; E-435 to Q-639; V-436 to Q-639; T-437 to Q-639; P-438 toQ-639; S-439 to Q-639; S-440 to Q-639; A-441 to Q-639; G-442 to Q-639;P-443 to Q-639; W-444 to Q-639; A-445 to Q-639; N-446 to Q-639; S-447 toQ-639; S-448 to Q-639; L-449 to Q-639; S-450 to Q-639; L-451 to Q-639;H-452 to Q-639; G-453 to Q-639; G-454 to Q-639; L-455 to Q-639; S-456 toQ-639; S-457 to Q-639; G-458 to Q-639; L-459 to Q-639; R-460 to Q-639;L-461 to Q-639; R-462 to Q-639; C-463 to Q-639; E-464 to Q-639; A-465 toQ-639; W-466 to Q-639; N-467 to Q-639; V-468 to Q-639; H-469 to Q-639;G-470 to Q-639; A-471 to Q-639; Q-472 to Q-639; S-473 to Q-639; G-474 toQ-639; S-475 to Q-639; I-476 to Q-639; L-477 to Q-639; Q-478 to Q-639;L-479 to Q-639; P-480 to Q-639; D-481 to Q-639; K-482 to Q-639; K-483 toQ-639; G-484 to Q-639; L-485to Q-639; I-486 to Q-639; S-487 to Q-639;T-488 to Q-639; A-489 to Q-639; F-490 to Q-639; S-491 to Q-639; N-492 toQ-639; G-493 to Q-639; A-494 to Q-639; F-495 to Q-639; L-496 to Q-639;G-497 to Q-639; 1498 to Q-639; G-499 to Q-639; I-500 to Q-639; T-501 toQ-639; A-502 to Q-639; L-503 to Q-639; L-504 to Q-639; F-505 to Q-639;L-506 to Q-639; C-507 to Q-639; L-508 to Q-639; A-509 to Q-639; L-510 toQ-639; I-511 to Q-639; I-512 to Q-639; M-513 to Q-639; K-514 to Q-639;I-515 to Q-639; L-516 to Q-639; P-517 to Q-639; K-518 to Q-639; R-519 toQ-639; R-520 to Q-639; T-521 to Q-639; Q-522 to Q-639; T-523 to Q-639;E-524 to Q-639; T-525 to Q-639; P-526 to Q-639; R-527 to Q-639; P-528toQ-639; R-529 to Q-639; F-530 to Q-639; S-531 to Q-639; R-532 to Q-639;H-533 to Q-639; S-534 to Q-639; T-535 to Q-639; I-536to Q-639; L-537 toQ-639; D-538 to Q-639; Y-539 to Q-639; I-540 to Q-639; N-541 to Q-639;V-542 to Q-639; V-543 to Q-639; P-544 to Q-639; T-545 to Q-639; A-546 toQ-639; G-547 to Q-639; P-548 to Q-639; L-549 to Q-639; A-550 to Q-639;Q-551 to Q-639; K-552 to Q-639; R-553 to Q-639; N-554 to Q-639; Q-555 toQ-639; K-556 to Q-639; A-557 to Q-639; T-558 to Q-639; P-559 to Q-639;N-560 to Q-639; S-561 to Q-639; P-562 to Q-639; R-563 to Q-639; T-564 toQ-639; P-565 to Q-639; L-566 to Q-639; P-567 to Q-639; P-568 to Q-639;G-569 to Q-639; A-570 to Q-639; P-571 to Q-639; S-572 to Q-639; P-573 toQ-639; E-574 to Q-639; S-575 to Q-639; K-576 to Q-639; K-577 to Q-639;N-578 to Q-639; Q-579 to Q-639; K-580 to Q-639; K-581 to Q-639; Q-582 toQ-639; Y-583 to Q-639; Q-584 to Q-639; L-585 to Q-639; P-586 to Q-639;S-587 to Q-639; F-588 to Q-639; P-589 to Q-639; E-590 to Q-639; P-591 toQ-639; K-592 to Q-639; S-593 to Q-639; S-594 to Q-639; T-595 to Q-639;Q-596 to Q-639; A-597 to Q-639; P-598 to Q-639; E-599 to Q-639; S-600 toQ-639; Q-601 to Q-639; E-602 to Q-639; S-603 to Q-639; Q-604 to Q-639;E-605 to Q-639; E-606 to Q-639; L-607 to Q-639; H-608 to Q-639; Y-609 toQ-639; A-610 to Q-639; T-611 to Q-639; L-612 to Q-639; N-613 to Q-639;F-614 to Q-639; P-615 to Q-639; G-616 to Q-639; V-617 to Q-639; R-618 toQ-639; P-619 to Q-639; R-620to Q-639; P-621 to Q-639; E-622 to Q-639;A-623 to Q-639; R-624 to Q-639; M-625 to Q-639; P-626 to Q-639; K-627 toQ-639; G-628 to Q-639; T-629 to Q-639; Q-630 to Q-639; A-631 to Q-639;D-632 to Q-639; Y-633 to Q-639; and A-634 to Q-639 of SEQ ID NO:33.Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g. fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities (e.g., ability to illicit mitogenicactivity, induce differentiation of normal or malignant cells, bind toEGF receptors, etc.)), may still be retained. For example the ability toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptide generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the C-terminus. Whether a particular polypeptide lackingC-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that a mutein with alarge number of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the polypeptide shown in FIGS. 13A-C, up to the leucineresidue at position number 7, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m1 of FIGS. 13A-C,where m1 is an integer from 7 to 638 corresponding to the position ofthe amino acid residue in FIGS. 13A-C. Moreover, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of C-terminal deletions of thepolypeptide of the invention shown as SEQ ID NO:33 include polypeptidescomprising, or alternatively consisting of, an amino acid sequenceselected from residues: M-1 to F-638; M-1 to K-637; M-1 to V-636; M-1 toE-635; M-1 to A-634; M-1 to Y-633; M-1 to D-632; M-1 to A-631; M-1 toQ-630; M-1 to T-629; M-1 to G-628; M-1 to K-627; M-1 to P-626; M-1 toM-625; M-1 to R-624; M-1 to A-623; M-1 to E-622; M-1 to P-621; M-1 toR-620; M-1 to P-619; M-1 to R-618; M-1 to V-617; M-1 to G-616; M-1 toP-615; M-1 to F-614; M-1 to N-613; M-1 to L-612; M-1 to T-611; M-1 toA-610; M-1 to Y-609; M-1 to H-608; M-1 to L-607; M-1 to E-606; M-1 toE-605; M-1 to Q-604; M-1 to S-603; M-1 to E-602; M-1 to Q-601; M-1 toS-600; M-1 to E-599; M-1 to P-598; M-1 to A-597; M-1 to Q-596; M-1 toT-595; M-1 to S-594; M-1 to S-593; M-1 to K-592; M-1 to P-591; M-1 toE-590; M-1 to P-589; M-1 to F-588; M-1 to S-587; M-1 to P-586; M-1 toL-585; M-1 to Q-584; M-1 to Y-583; M-1 to Q-582; M-1 to K-581; M-1 toK-580; M-1 to Q-579; M-1 to N-578; M-1 to K-577; M-1 to K-576; M-1 toS-575; M-1 to E-574; M-1 to P-573; M-1 to S-572; M-1 to P-571; M-1 toA-570; M-1 to G-569; M-1 to P-568; M-1 to P-567; M-1 to L-566; M-1 toP-565; M-1 to T-564; M-1 to R-563; M-1 to P-562; M-1 to S-561; M-1 toN-560; M-1 to P-559; M-1 to T-558; M-1 to A-557; M-1 to K-556; M-1 toQ-555; M-1 to N-554; M-1 to R-553; M-1 to K-552; M-1 to Q-551; M-1 toA-550; M-1 to L-549; M-1 to P-548; M-1 to G-547; M-1 A-546; M-1 toT-545; M-1 to P-544; M-1 to V-543; M-1 to V-542; M-1 to N-541; M-1 toI-540; M-1 to Y-539; M-1 to D-538; M-1 to L-537; M-1 to I-536; M-1 toT-535; M-1 to S-534; M-1 to H-533; M-1 to R-532; M-1 to S-531; M-1 toF-530; M-1 to R-529; M-1 to P-528; M-1 to R-527; M-1 to P-526; M-1 toT-525; M-1 to E-524; M-1 to T-523; M-1 to Q-522; M-1 to T-521; M-1 toR-520; M-1 to R-519; M-1 to K-518; M-1 to P-517; M-1 to L-516; M-1 toI-515; M-1 to K-514; M-1 to M-513; M-1 to I-512; M-1 to I-511; M-1 toL-510; M-1 to A-509; M-1 to L-508; M-1 to C-507; M-1 to L-506; M-1 toF-505; M-1 to L-504; M-1 to L-503; M-1 to A-502; M-1 to T-501; M-1 toI-500; M-1 to G-499; M-1 to I-498; M-1 to G-497; M-1 to L-496; M-1 toF-495; M-1 to A-494; M-1 to G-493; M-1 to N-492; M-1 to S-491; M-1 toF-490; M-1 to A-489; M-1 to T-488; M-1 to S-487; M-1 to I-486; M-1 toL-485; M-1 to G-484; M-1 to K-483; M-1 to K-482; M-1 to D-481; M-1 toP-480; M-1 to L-479; M-1 to Q-478; M-1 to L-477; M-1 to I-476; M-1 toS-475; M-1 to G-474; M-1 to S-473; M-1 to Q-472; M-1 to A-471; M-1 toG-470; M-1 to H-469; M-1 to V-468; M-1 to N-467; M-1 to W-466; M-1 toA-465; M-1 to E-464; M-1 to C-463; M-1 to R-462; M-1 to L-461; M-1 toR-460; M-1 to L-459; M-1 to G-458; M-1 to S-457; M-1 to S-456; M-1 toL-455; M-1 to G-454; M-1 to G-453; M-1 to H-452; M-1 to L-451; M-1 toS-450; M-1 to L-449; M-1 to S-448; M-1 to S-447; M-1 to N-446; M-1 toA-445; M-1 to W-444; M-1 to P-443; M-1 to G-442; M-1 to A-441; M-1 toS-440; M-1 to S-439; M-1 to P-438; M-1 to T-437; M-1 to V-436; M-1 toE-435; M-1 to F-434; M-1 to S-433; M-1 to D-432; M-1 to Q-431; M-1 toS-430; M-1 to S-429; M-1 to N-428; M-1 to G-427; M-1 to E-426; M-1 toL-425; M-1 to L-424; M-1 to E-423; M-1 to E-422; M-1 to G-421; M-1 toL-420; M-1 to W-419; M-1 to W-418; M-1 to R-417; M-1 to L-416; M-1 toS-415; M-1 to P-414; M-1 to A-413; M-1 to P-412; M-1 to S-411; M-1 toA-410; M-1 to Q-409; M-1 to S-408; M-1 to S-407; M-1 to C-406; M-1 toS-405; M-1 to C-404; M-1 to H-403; M-1 to L-402; M-1 to G-401; M-1 toE-400; M-1 to A-399; M-1 to E-398; M-1 to W-397; M-1 to S-396; M-1 toC-395; M-1 to S-394; M-1 to P-393; M-1 to G-392; M-1 to L-391; M-1 toL-390; M-1 to K-389; M-1 to P-388; M-1 to S-387; M-1 to Y-386; M-1 toH-385; M-1 to V-384; M-1 to S-383; M-1 to L-382; M-1 to S-381; M-1 toL-380; M-1 to S-379; M-1 to V-378; M-1 to H-377; M-1 to Q-376; M-1 toS-375; M-1 to G-374; M-1 to L-373; M-1 to P-372; M-1 to H-371; M-1 toR-370; M-1 to A-369; M-1 to H-368; M-1 to C-367; M-1 to T-366; M-1 toF-365; M-1 to E-364; M-1 to G-363; M-1 to E-362; M-1 to H-361; M-1 toE-360; M-1 to V-359; M-1 to Q-358; M-1 to V-357; M-1 to R-356; M-1 toP-355; M-1 to L-354; M-1 to E-353; M-1 to L-352; M-1 to V-351; M-1 toG-350; M-1 to P-349; M-1 to D-348; M-1 to S-347; M-1 to P-346; M-1 toQ-345; M-1 to S-344; M-1 to P-343; M-1 to S-342; M-1 to L-341; M-1 toV-340; M-1 to Q-339; M-1 to G-338; M-1 to R-337; M-1 to Q-336; M-1 toT-335; M-1 to W-334; M-1 to S-333; M-1 to L-332; M-1 to R-331; M-1 toA-330; M-1 to P-329; M-1 to P-328; M-1 to S-327; M-1 to S-326; M-1 toH-325; M-1 to T-324; M-1 to V-323; M-1 to C-322; M-1 to V-321; M-1 toL-320; M-1 to C-319; M-1 to L-318; M-1 to S-317; M-1 to Q-316; M-1 toG-315; M-1 to E-314; M-1 to L-313; M-1 to V-312; M-1 to P-311; M-1 toL-310; M-1 to S-309; M-1 to T-308; M-1 to G-307; M-1 to N-306; M-1 toG-305; M-1 to L-304; M-1 to N-303; M-1 to E-302; M-1 to L-301; M-1 toV-300; M-1 to T-299; M-1 to R-298; M-1 to N-297; M-1 to A-296; M-1 toQ-295; M-1 to S-294; M-1 to V-293; M-1 to M-292; M-1 to V-291; M-1 toR-290; M-1 to L-289; M-1 to N-288; M-1 to E-287; M-1 to P-286; M-1 toP-285; M-1 to Y-284; M-1 to Q-283; M-1 to V-282; M-1 to S-281; M-1 toL-280; M-1 to D-279; M-1 to L-278; M-1 to A-277; M-1 to R-276; M-1 toQ-275; M-1 to Q-274; M-1 to S-273; M-1 to G-272; M-1 to L-271; M-1 toR-270; M-1 to N-269; M-1 to E-268; M-1 to A-267; M-1 to R-266; M-1 toC-265; M-1 to T-264; M-1 to Y-263; M-1 to R-262; M-1 to G-261; M-1 toS-260; M-1 to D-259; M-1 to G-258; M-1 to A-257; M-1 to K-256; M-1 toV-255; M-1 to G-254; M-1 to P-253; M-1 to L-252; M-1 to E-251; M-1 toL-250; M-1 to G-249; M-1 to L-248; M-1 to P-247; M-1 to R-246; M-1 toP-245; M-1 to G-244; M-1 to W-243; M-1 to P-242; M-1 to H-241; M-1 toS-240; M-1 to S-239; M-1 to S-238; M-1 to L-237; M-1 to V-236; M-1 toR-235; M-1 to N-234; M-1 to Q-233; M-1 to L-232; M-1 to V-231; M-1 toW-230; M-1 to S-229; M-1 to L-228; M-1 to T-277; M-1 to A-226; M-1 toP-225; M-1 to P-224; M-1 to Q-223; M-1 to S-222; M-1 to D-221; M-1 toA-220; M-1 to A-219; M-1 to C-218; M-1 to L-217; M-1 to L-216; M-1 toR-215; M-1 to L-214; M-1 to F-213; M-1 to Q-212; M-1 to G-211; M-1 toK-210; M-1 to Q-209; M-1 to A-208; M-1 to E-207; M-1 to L-206; M-1 toY-205; M-1 to P-204; M-1 to V-203; M-1 to N-202; M-1 to G-201; M-1 toQ-200; M-1 to P-199; M-1 to Q-198; M-1 to P-197; M-1 to E-196; M-1 toL-195; M-1 to A-194; M-1 to P-193; M-1 to T-192; M-1 to N-191; M-1 toD-190; M-1 to R-189; M-1 to S-188; M-1 to I-187; M-1 to S-186; M-1 toI-185; M-1 to V-184; M-1 to L-183; M-1 to D-182; M-1 to R-181; M-1 toP-180; M-1 to A-179; M-1 to Y-178; M-1 to A-177; M-1 to V-176; M-1 toR-175; M-1 to L-174; M-1 to R-173; M-1 to V-172; M-1 to T-171; M-1 toR-170; M-1 to Q-169; M-1 to A-168; M-1 to S-167; M-1 to V-166; M-1 toG-165; M-1 to K-164; M-1 to R-163; M-1 to S-162; M-1 to F-161; M-1 toD-160; M-1 to V-159; M-1 to H-158; M-1 to C-157; M-1 to T-156; M-1 toL-155; M-1 to D-154; M-1 to T-153; M-1 to N-152; M-1 to H-151; M-1 toD-150; M-1 to Q-149; M-1 to P-148; M-1 to R-147; M-1 to P-146; M-1 toT-145; M-1 to F-144; M-1 to S-143; M-1 to L-142; M-1 to V-141; M-1 toT-140; M-1 to V-139; M-1 to K-138; M-1 to L-137; M-1 to F-136; M-1 toF-135; M-1 to G-134; M-1 to D-133; M-1 to N-132; M-1 to M-131; M-1 toF-130; M-1 to N-129; M-1 to Y-128; M-1 to R-127; M-1 to V-126; M-1 toY-125; M-1 to S-124; M-1 to G-123; M-1 to R-122; M-1 to E-121; M-1 toV-120; M-1 to R-119; M-1 to F-118; M-1 to F-117; M-1 to Y-116; M-1 toQ-115; M-1 to S-114; M-1 to E-113; M-1 to D-112; M-1 to Q-111; M-1 toM-110; M-1 to Q-109; M-1 to A-108; M-1 to D-107; M-1 to R-106; M-1 toI-105; M-1 to V-104; M-1 to L-103; M-1 to S-102; M-1 to C-101; M-1 toN-100; M-1 to G-99; M-1 to K-98; M-1 to A-97; M-1 to P-96; M-1 to D-95;M-1 to G-94; M-1 to T-93; M-1 to L-92; M-1 to Q-91; M-1 to F-90; M-1 toR-89; M-1 to G-88; M-1 to R-87; M-1 to T-86; M-1 to S-85; M-1 to M-84;M-1 to E-83; M-1 to V-82; M-1 to E-81; M-1 to R-80; M-1 to S-79; M-1 toQ-78; M-1 to H-77; M-1 to N-76; M-1 to T-75; M-1 to A-74; M-1 to V-73;M-1 to P-72; M-1 to A-71; M-1 to G-70; M-1 to K-69; M-1 to T-68; M-1 toT-67; M-1 to E-66; M-1 to T-65; M-1 to V-64; M-1 to A-63; M-1 to K-62;M-1 to F-61; M-1 to W-60; M-1 to Y-59; M-1 to G-58; M-1 to Y-57; M-1 toA-56; M-1 to P-55; M-1 to T-54; M-1 to S-53; M-1 to G-52; M-1 to T-51;M-1 to W-50; M-1 to D-49; M-1 to Q-48; M-1 to R-47; M-1 to P-46; M-1 toY-45; M-1 to S-44; M-1 to F-43; M-1 to S-42; M-1 to C-41; M-1 to P-40;M-1 to V-39; M-1 to S-38; M-1 to I-37; M-1 to D-36; M-1 to C-35; M-1 toA-34; M-1 to E-33; M-1 to P-32; M-1 to V-31; M-1 to M-30; M-1 to V-29;M-1 to S-28; M-1 to E-27; M-1 to Q-26; M-1 to V-25; M-1 to R-24; M-1 toI-23; M-1 to W-22; M-1 to F-21; M-1 to R-20; M-1 to G-19; M-1 to D-18;M-1 to M-17; M-1 to A-16; M-1 to Q-15; M-1 to S-14; M-1 to G-13; M-1 toG-12; M-1 to L-11; M-1 to L-10; M-1 to S-9; M-1 to S-8; and M-1 to L-7of SEQ ID NO:33. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g. fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

Further preferred polypeptides of the invention include N-terminaldeletions of CD33-likeSV (SEQ ID NO: 149), these deletions comprising,or alternatively consisting of, an amino acid sequence selected fromresidues: L-2 to Q-697; L-3 to Q-697; P-4 to Q-697; L-5 to Q-697; L-6 toQ-697; L-7 to Q-697; S-8 to Q-697; S-9 to Q-697; L-10 to Q-697; L-11 toQ-697; G-12 to Q-697; G-13 to Q-697; S-14 to Q-697; Q-15 to Q-697; A-16to Q-697; M-17 to Q-697; D-18 to Q-697; G-19 to Q-697; R-20 to Q-697;F-21 to Q-697; W-22 to Q-697; I-23 to Q-697; R-24 to Q-697; V-25 toQ-697; Q-26 to Q-697; E-27 to Q-697; S-28 to Q-697; V-29 to Q-697; M-30to Q-697; V-31 to Q-697; P-32 to Q-697; E-33 to Q-697; G-34 to Q-697;L-35 to Q-697; C-36 to Q-697; I-37 to Q-697; S-38 to Q-697; V-39 toQ-697; P-40 to Q-697; C-41 to Q-697; S-42 to Q-697; F-43 to Q-697; S-44to Q-697; Y-45 to Q-697; P-46 to Q-697; R-47 to Q-697; Q-48 to Q-697;D-49 to Q-697; W-50 to Q-697; T-51 to Q-697; G-52 to Q-697; S-53 toQ-697; T-54 to Q-697; P-55 to Q-697; A-56 to Q-697; Y-57 to Q-697; G-58to Q-697; Y-59 to Q-697; W-60 to Q-697; F-61 to Q-697; K-62 to Q-697;A-63 to Q-697; V-64 to Q-697; T-65 to Q-697; E-66 to Q-697; T-67 toQ-697; T-68 to Q-697; K-69 to Q-697; G-70 to Q-697; A-71 to Q-697; P-72to Q-697; V-73 to Q-697; A-74 to Q-697; T-75 to Q-697; N-76 to Q-697;H-77 to Q-697; Q-78 to Q-697; S-79 to Q-697; R-80 to Q-697; E-81 toQ-697; V-82 to Q-697; E-83 to Q-697; M-84 to Q-697; S-85 to Q-697; T-86to Q-697; R-87 to Q-697; G-88 to Q-697; R-89 to Q-697; F-90 to Q-697;Q-91 to Q-697; L-92 to Q-697; T-93 to Q-697; G-94 to Q-697; D-95 toQ-697; P-96 to Q-697; A-97 to Q-697; K-98 to Q-697; G-99 to Q-697; N-100to Q-697; C-101 to Q-697; S-102 to Q-697; L-103 to Q-697; V-104 toQ-697; I-105 to Q-697; R-106 to Q-697; D-107 to Q-697; A-108 to Q-697;Q-109 to Q-697; M-110 to Q-697; Q-111 to Q-697; D-112 to Q-697; E-113 toQ-697; S-114 to Q-697; Q-115 to Q-697; Y-116 to Q-697; F-117 to Q-697;F-118 to Q-697; R-119 to Q-697; V-120 to Q-697; E-121 to Q-697; R-122 toQ-697; G-123 to Q-697; S-124 to Q-697; Y-125 to Q-697; V-126 to Q-697;R-127 to Q-697; Y-128 to Q-697; N-129 to Q-697; F-130 to Q-697; M-131 toQ-697; N-132 to Q-697; D-133 to Q-697; G-134 to Q-697; F-135 to Q-697;F-136 to Q-697; L-137 to Q-697; K-138 to Q-697; V-139 to Q-697; T-140 toQ-697; A-141 to Q-697; L-142 to Q-697; T-143 to Q-697; Q-144 to Q-697;K-145 to Q-697; P-146 to Q-697; D-147 to Q-697; V-148 to Q-697; Y-149 toQ-697; I-150 to Q-697; P-151 to Q-697; E-152 to Q-697; T-153 to Q-697;L-154 to Q-697; E-155 to Q-697; P-156 to Q-697; G-157 to Q-697; Q-158 toQ-697; P-159 to Q-697; V-160 to Q-697; T-161 to Q-697; V-162 to Q-697;I-163 to Q-697; C-164 to Q-697; V-165 to Q-697; F-166 to Q-697; N-167 toQ-697; W-168 to Q-697; A-169 to Q-697; F-170 to Q-697; E-171 to Q-697;E-172 to Q-697; C-173 to Q-697; P-174 to Q-697; P-175 to Q-697; P-176 toQ-697; S-177 to Q-697; F-178 to Q-697; S-179 to Q-697; W-180 to Q-697;T-181 to Q-697; G-182 to Q-697; A-183 to Q-697; A-184 to Q-697; L-185 toQ-697; S-186 to Q-697; S-187 to Q-697; Q-188 to Q-697; G-189 to Q-697;T-190 to Q-697; K-191 to Q-697; P-192 to Q-697; T-193 to Q-697; T-194 toQ-697; S-195 to Q-697; H-196 to Q-697; F-197 to Q-697; S-198 to Q-697;V-199 to Q-697; L-200 to Q-697; S-201 to Q-697; F-202 to Q-697; T-203 toQ-697; P-204 to Q-697; R-205 to Q-697; P-206 to Q-697; Q-207 to Q-697;D-208 to Q-697; H-209 to Q-697; N-210 to Q-697; T-211 to Q-697; D-212 toQ-697; L-213 to Q-697; T-214 to Q-697; C-215 to Q-697; H-216 to Q-697;V-217 to Q-697; D-218 to Q-697; F-219 to Q-697; S-220 to Q-697; R-221 toQ-697; K-222 to Q-697; G-223 to Q-697; V-224 to Q-697; S-224 to Q-697;S-225 to Q-697; V-226 to Q-697; Q-227 to Q-697; R-228 to Q-697; T-229 toQ-697; V-230 to Q-697; R-231 to Q-697; L-232 to Q-697; R-233 to Q-697;V-234 to Q-697; A-235 to Q-697; Y-236 to Q-697; A-237 to Q-697; P-238 toQ-697; R-239 to Q-697; D-240 to Q-697; L-241 to Q-697; V-242 to Q-697;I-243 to Q-697; S-244 to Q-697; I-245 to Q-697; S-246 to Q-697; R-247 toQ-697; D-248 to Q-697; N-249 to Q-697; T-250 to Q-697; P-251 to Q-697;A-252 to Q-697; L-253 to Q-697; E-254 to Q-697; P-255 to Q-697; Q-256 toQ-697; P-257 to Q-697; Q-258 to Q-697; G-259 to Q-697; N-260 to Q-697;V-261 to Q-697; P-262 to Q-697; Y-263 to Q-697; L-264 to Q-697; E-265 toQ-697; A-266 to Q-697; Q-267 to Q-697; K-268 to Q-697; G-269 to Q-697;Q-270 to Q-697; F-271 to Q-697; L-272 to Q-697; R-273 to Q-697; L-274 toQ-697; L-275 to Q-697; C-276 to Q-697; A-277 to Q-697; A-278 to Q-697;D-279 to Q-697; S-280 to Q-697; Q-281 to Q-697; P-282 to Q-697; P-283 toQ-697; A-284 to Q-697; T-285 to Q-697; L-286 to Q-697; S-287 to Q-697;W-288 to Q-697; V-289 to Q-697; L-290 to Q-697; Q-291 to Q-697; N-292 toQ-697; R-293 to Q-697; V-294 to Q-697; L-295 to Q-697; S-296 to Q-697;S-297 to Q-697; S-298 to Q-697; H-299 to Q-697; P-300 to Q-697; W-301 toQ-697; G-302 to Q-697; P-303 to Q-697; R-304 to Q-697; P-305 to Q-697;L-306 to Q-697; G-307 to Q-697; L-308 to Q-697; E-309 to Q-697; L-310 toQ-697; P-311 to Q-697; G-312 to Q-697; V-313 to Q-697; K-314 to Q-697;A-315 to Q-697; G-316 to Q-697; D-317 to Q-697; S-318 to Q-697; G-319 toQ-697; R-320 to Q-697; Y-321 to Q-697; T-322 to Q-697; C-323 to Q-697;R-324 to Q-697; A-325 to Q-697; E-326 to Q-697; N-327 to Q-697; R-328 toQ-697; L-329 to Q-697; G-330 to Q-697; S-331 to Q-697; Q-332 to Q-697;Q-333 to Q-697; R-334 to Q-697; A-335 to Q-697; L-336 to Q-697; D-337 toQ-697; L-338 to Q-697; S-339 to Q-697; V-340 to Q-697; Q-341 to Q-697;Y-342 to Q-697; P-343 to Q-697; P-344 to Q-697; E-345 to Q-697; N-346 toQ-697; L-347 to Q-697; R-348 to Q-697; V-349 to Q-697; M-350 to Q-697;V-351 to Q-697; S-352 to Q-697; Q-353 to Q-697; A-354 to Q-697; N-355 toQ-697; R-356 to Q-697; T-357 to Q-697; V-358 to Q-697; L-359 to Q-697;E-360 to Q-697; N-361 to Q-697; L-362 to Q-697; G-363 to Q-697; N-364 toQ-697; G-365 to Q-697; T-366 to Q-697; S-367 to Q-697; L-368 to Q-697;P-369 to Q-697; V-370 to Q-697; L-371 to Q-697; E-372 to Q-697; G-373 toQ-697; Q-374 to Q-697; S-375 to Q-697; L-376 to Q-697; C-377 to Q-697;L-378 to Q-697; V-379 to Q-697; C-380 to Q-697; V-381 to Q-697; T-382 toQ-697; H-383 to Q-697; S-384 to Q-697; S-385 to Q-697; P-386 to Q-697;P-387 to Q-697; A-388 to Q-697; R-389 to Q-697; L-390 to Q-697; S-391 toQ-697; W-392 to Q-697; T-393 to Q-697; Q-394 to Q-697; R-395 to Q-697;G-396 to Q-697; Q-397 to Q-697; V-398 to Q-697; L-399 to Q-697; S-400 toQ-697; P-401 to Q-697; S-402 to Q-697; Q-403 to Q-697; P-404 to Q-697;S-405 to Q-697; D-406 to Q-697; P-407 to Q-697; G-408 to Q-697; V-409 toQ-697; L-410 to Q-697; E-411 to Q-697; L-412 to Q-697; P-413 to Q-697;R-414 to Q-697; V-415 to Q-697; Q-416 to Q-697; V-417 to Q-697; E-418 toQ-697; H-419 to Q-697; E-420 to Q-697; G-421 to Q-697; E-422 to Q-697;F-423 to Q-697; T-424 to Q-697; C-425 to Q-697; H-426 to Q-697; A-427 toQ-697; R-428 to Q-697; H-429 to Q-697; P-430 to Q-697; L-431 to Q-697;G-432 to Q-697; S-433 to Q-697; Q-434 to Q-697; H-435 to Q-697; V-436 toQ-697; S-437 to Q-697; L-438 to Q-697; S-439 to Q-697; L-440 to Q-697;S-441 to Q-697; V-442 to Q-697; H-443 to Q-697; Y-444 to Q-697; S-445 toQ-697; P-446 to Q-697; K-447 to Q-697; L-448 to Q-697; L-449 to Q-697;G-450 to Q-697; P-451 to Q-697; S-452 to Q-697; C-453 to Q-697; S-454 toQ-697; W-455 to Q-697; E-456 to Q-697; A-457 to Q-697; E-458 to Q-697;G-459 to Q-697; L-460 to Q-697; H-461 to Q-697; C-462 to Q-697; S-463 toQ-697; C-464 to Q-697; S-465 to Q-697; S-466 to Q-697; Q-467 to Q-697;A-468 to Q-697; S-469 to Q-697; P-470 to Q-697; A-471 to Q-697; P-472 toQ-697; S-473 to Q-697; L-474 to Q-697; R-475 to Q-697; W-476 to Q-697;W-477 to Q-697; L-478 to Q-697; G-479 to Q-697; E-480 to Q-697; E-481 toQ-697; L-482 to Q-697; L-483 to Q-697; E-484 to Q-697; G-485 to Q-697;N-486 to Q-697; S-487 to Q-697; S-488 to Q-697; Q-489 to Q-697; D-490 toQ-697; S-491 to Q-697; F-492 to Q-697; E-493 to Q-697; V-494 to Q-697;T-495 to Q-697; P-496 to Q-697; S-497 to Q-697; S-498 to Q-697; A-499 toQ-697; G-500 to Q-697; P-501 to Q-697; W-502 to Q-697; A-503 to Q-697;N-504 to Q-697; S-505 to Q-697; S-506 to Q-697; L-507 to Q-697; S-508 toQ-697; L-509 to Q-697; H-510 to Q-697; G-511 to Q-697; G-512 to Q-697;L-513 to Q-697; S-514 to Q-697; S-515 to Q-697; G-516 to Q-697; L-517 toQ-697; R-518 to Q-697; L-519 to Q-697; R-520 to Q-697; C-521 to Q-697;E-522 to Q-697; A-523 to Q-697; W-524 to Q-697; N-525 to Q-697; V-526 toQ-697; H-527 Q-697; G-528 to Q-697; A-529 to Q-697; Q-530 to Q-697;S-531 to Q-697; G-532 to Q-697; S-533 to Q-697; I-534 to Q-697; L-535 toQ-697; Q-536 to Q-697; L-537 to Q-697; P-538 to Q-697; D-539 to Q-697;K-540 to Q-697; K-541 to Q-697; G-542 to Q-697; L-543 to Q-697; I-544 toQ-697; S-545 to Q-697; T-546 to Q-697; A-547 to Q-697; F-548 to Q-697;S-549 to Q-697; N-550 to Q-697; G-551 to Q-697; A-552 to Q-697; F-553 toQ-697; L-554 to Q-697; G-555 to Q-697; I-556 to Q-697; G-557 to Q-697;I-558 to Q-697; T-559 to Q-697; A-560 to Q-697; L-561 to Q-697; L-562 toQ-697; F-563 to Q-697; L-564 to Q-697; C-565 to Q-697; L-566 to Q-697;A-567 to Q-697; L-568 to Q-697; I-569 to Q-697; I-570 to Q-697; M-571 toQ-697; K-572 to Q-697; I-573 to Q-697; L-574 to Q-697; P-575 to Q-697;K-576 to Q-697; R-577 to Q-697; R-578 to Q-697; T-579 to Q-697; Q-580 toQ-697; T-581 to Q-697; E-582 to Q-697; T-583 to Q-697; P-584 to Q-697;R-585 to Q-697; P-586 to Q-697; R-587 to Q-697; F-588 to Q-697; S-589 toQ-697; R-590 to Q-697; H-591 to Q-697; S-592 to Q-697; T-593 to Q-697;I-594 to Q-697; L-595 to Q-697; D-596 to Q-697; Y-597 to Q-697; I-598 toQ-697; N-599 to Q-697; V-600 to Q-697; V-601 to Q-697; P-602 to Q-697;T-603 to Q-697; A-604 to Q-697; G-605 to Q-697; P-606 to Q-697; L-607 toQ-697; A-608 to Q-697; Q-609 to Q-697; K-610 to Q-697; R-611 to Q-697;N-612 to Q-697; Q-613 to Q-697; K-614 to Q-697; A-615 to Q-697; T-616 toQ-697; P-617 to Q-697; N-618 to Q-697; S-619 to Q-697; P-620 to Q-697;R-621 to Q-697; T-622 to Q-697; P-623 to Q-697; L-624 to Q-697; P-625 toQ-697; P-626 to Q-697; G-627 to Q-697; A-628 to Q-697; P-629 to Q-697;S-630 to Q-697; P-631 to Q-697; E-632 to Q-697; S-633 to Q-697; K-634 toQ-697; K-635 to Q-697; N-636 to Q-697; Q-637 to Q-697; K-638 to Q-697;K-639 to Q-697; Q-640 to Q-697; Y-641 to Q-697; Q-642 to Q-697; L-643 toQ-697; P-644 to Q-697; S-645 to Q-697; F-646 to Q-697; P-647 to Q-697;E-648 to Q-697; P-649 to Q-697; K-650 to Q-697; S-651 to Q-697; S-652 toQ-697; T-653 to Q-697; Q-654 to Q-697; A-655 to Q-697; P-656 to Q-697;E-657 to Q-697; S-658 to Q-697; Q-659 to Q-697; E-660 to Q-697; S-661 toQ-697; Q-662 to Q-697; E-663 to Q-697; E-664 to Q-697; L-665 to Q-697;H-666 to Q-697; Y-667 to Q-697; A-668 to Q-697; T-669 to Q-697; L-670 toQ-697; N-671 to Q-697; F-672 to Q-697; P-673 to Q-697; G-674 to Q-697;V-675 to Q-697; R-676 to Q-697; P-677 to Q-697; R-678 to Q-697; P-679 toQ-697; E-680 to Q-697; A-681 to Q-697; R-682 to Q-697; M-683 to Q-697;P-684 to Q-697; K-685 to Q-697; G-686 to Q-697; T-687 to Q-697; Q-688 toQ-697; A-689 to Q-697; D-690 to Q-697; Y-691 to Q-697; A-692 to Q-697 ofSEQ ID NO: 149. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g. fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

Moreover, the invention provides C-terminal amino acid deletions ofCD33-likeSV (SEQ ID NO: 149) comprising, or alternatively consisting of,an amino acid sequence selected from residues: M-1 to F-696; M-1 toK-695; M-1 to V-694; M-1 to E-693; M-1 to A-692; M-1 to Y-691; M-1 toD-690; M-1 to A-689; M-1 to Q-688; M-1 to T-687; M-1 to G-686; M-1 toK-685; M-1 to P-684; M-1 to M-683; M-1 to R-682; M-1 to A-681; M-1 toE-680; M-1 to P-679; M-1 to R-678; M-1 to P-677; M-1 to R-676; M-1 toV-675; M-1 to G-674; M-1 to P-673; M-1 to F-672; M-1 to N-671; M-1 toL-670; M-1 to T-669; M-1 to A-668; M-1 to Y-667; M-1 to H-666; M-1 toL-665; M-1 to E-664; M-1 to E-663; M-1 to Q-662; M-1 to S-661; M-1 toE-660; M-1 to Q-659; M-1 to S-658; M-1 to E-657; M-1 to P-656; M-1 toA-655; M-1 to Q-654; M-1 to T-653; M-1 to S-652; M-1 to S-651; M-1 toK-650; M-1 to P-649; M-1 to E-648; M-1 to P-647; M-1 to F-646; M-1 toS-645; M-1 to P-644; M-1 to L-643; M-1 to Q-642; M-1 to Y-641; M-1 toQ-640; M-1 to K-639; M-1 to K-638; M-1 to Q-637; M-1 to N-636; M-1 toK-635; M-1 to K-634; M-1 to S-633; M-1 to E-632; M-1 to P-631; M-1 toS-630; M-1 to P-629; M-1 to A-628; M-1 to G-627; M-1 to P-626; M-1 toP-625; M-1 to L-624; M-1 to P-623; M-1 to T-622; M-1 to R-621; M-1 toP-620; M-1 to S-619; M-1 to N-618; M-1 to P-617; M-1 to T-616; M-1 toA-615; M-1 to K-614; M-1 to Q-613; M-1 to N-612; M-1 to R-611; M-1 toK-610; M-1 to Q-609; M-1 to A-608; M-1 to L-607; M-1 to P-606; M-1 toG-605; M-1 to A-604; M-1 to T-603; M-1 to P-602; M-1 to V-601; M-1 toV-600; M-1 to N-599; M-1 to I-598; M-1 to Y-597; M-1 to D-596; M-1 toL-595; M-1 to I-594; M-1 to T-593; M-1 to S-592; M-1 to H-591; M-1 toR-590; M-1 to S-589; M-1 to F-588; M-1 to R-587; M-1 to P-586; M-1 toR-585; M-1 to P-584; M-1 to T-583; M-1 to E-582; M-1 to T-581; M-1 toQ-580; M-1 to T-579; M-1 to R-578; M-1 to R-577; M-1 to K-576; M-1 toP-575; M-1 to L-574; M-1 to I-573; M-1 to K-572; M-1 to M-571; M-1 toI-570; M-1 to I-569; M-1 to L-568; M-1 to A-567; M-1 to L-566; M-1 toC-565; M-1 to L-564; M-1 to F-563; M-1 to L-562; M-1 to L-561; M-1 toA-560; M-1 to T-559; M-1 to I-558; M-1 to G-557; M-1 to I-556; M-1 toG-555; M-1 to L-554; M-1 to F-553; M-1 to A-552; M-1 to G-551; M-1 toN-550; M-1 to S-549; M-1 to F-548; M-1 to A-547; M-1 to T-546; M-1 toS-545; M-1 to I-544; M-1 to L-543; M-1 to G-542; M-1 to K-541; M-1 toK-540; M-1 to D-539; M-1 to P-538; M-1 to L-537; M-1 to Q-536; M-1 toL-535; M-1 to I-534; M-1 to S-533; M-1 to G-532; M-1 to S-531; M-1 toQ-530; M-1 to A-529; M-1 to G-528; M-1 to H-527; M-1 to V-526; M-1 toN-525; M-1 to W-524; M-1 to A-523; M-1 to E-522; M-1 to C-521; M-1 toR-520; M-1 to L-519; M-1 to R-518; M-1 to L-517; M-1 to G-516; M-1 toS-515; M-1 to S-514; M-1 to L-513; M-1 to G-512; M-1 to G-511; M-1 toH-510; M-1 to L-509; M-1 to S-508; M-1 to L-507; M-1 to S-506; M-1 toS-505; M-1 to N-504; M-1 to A-503; M-1 to W-502; M-1 to P-501; M-1 toG-500; M-1 to A-499; M-1 to S-498; M-1 to S-497; M-1 to P-496; M-1 toT495; M-1 to V-494; M-1 to E-493; M-1 to F-492; M-1 to S-491; M-1 toD-490; M-1 to Q-489; M-1 to S-488; M-1 to S-487; M-1 to N-486; M-1 toG-485; M-1 to E-484; M-1 to L-483; M-1 to L-482; M-1 to E-481; M-1 toE-480; M-1 to G-479; M-1 to L-478; M-1 to W-477; M-1 to W-476; M-1 toR-475; M-1 to L-474; M-1 to S-473; M-1 to P-472; M-1 to A-471; M-1 toP-470; M-1 to S-469; M-1 to A-468; M-1 to Q-467; M-1 to S-466; M-1 toS-465; M-1 to C-464; M-1 to S-463; M-1 to C-462; M-1 to H-461; M-1 toL-460; M-1 to G-459; M-1 to E-458; M-1 to A-457; M-1 to E-456; M-1 toW-455; M-1 to S-454; M-1 to C-453; M-1 to S-452; M-1 to P-451; M-1 toG-450; M-1 to L-449; M-1 to L-448; M-1 to K-447; M-1 to P-446; M-1 toS-445; M-1 to Y-444; M-1 to H-443; M-1 to V-442; M-1 to S-441; M-1 toL-440; M-1 to S-439; M-1 to L-438; M-1 to S-437; M-1 to V-436; M-1 toH-435; M-1 to Q-434; M-1 to S-433; M-1 to G-432; M-1 to L-431; M-1 toP-430; M-1 to H-429; M-1 to R-428; M-1 to A-427; M-1 to H-426; M-1 toC-425; M-1 to T-424; M-1 to F-423; M-1 to E-422; M-1 to G-421; M-1 toE-420; M-1 to H-419; M-1 to E-418; M-1 to V-417; M-1 to Q-416; M-1 toV-415; M-1 to R-414; M-1 to P-413; M-1 to L-412; M-1 to E-411; M-1 toL-410; M-1 to V-409; M-1 to G-408; M-1 to P-407; M-1 to D-406; M-1 toS-405; M-1 to P-404; M-1 to Q-403; M-1 to S-402; M-1 to P-401; M-1 toS-400; M-1 to L-399; M-1 to V-398; M-1 to Q-397; M-1 to G-396; M-1 toR-395; M-1 to Q-394; M-1 to T-393; M-1 to W-392; M-1 to S-391; M-1 toL-390; M-1 to R-389; M-1 to A-388; M-1 to P-387; M-1 to P-386; M-1 toS-385; M-1 to S-384; M-1 to H-383; M-1 to T-382; M-1 to V-381; M-1 toC-380; M-1 to V-379; M-1 to L-378; M-1 to C-377; M-1 to L-376; M-1 toS-375; M-1 to Q-374; M-1 to G-373; M-1 to E-372; M-1 to L-371; M-1 toV-370; M-1 to P-369; M-1 to L-368; M-1 to S-367; M-1 to T-366; M-1 toG-365; M-1 to N-364; M-1 to G-363; M-1 to L-362; M-1 to N-361; M-1 toE-360; M-1 to L-359; M-1 to V-358; M-1 to T-357; M-1 to R-356; M-1 toN-355; M-1 to A-354; M-1 to Q-353; M-1 to S-352; M-1 to V-351; M-1 toM-350; M-1 to V-349; M-1 to R-348; M-1 to L-347; M-1 to N-346; M-1 toE-345; M-1 to P-344; M-1 to P-343; M-1 to Y-342; M-1 to Q-341; M-1 toV-340; M-1 to S-339; M-1 to L-338; M-1 to D-337; M-1 to L-336; M-1 toA-335; M-1 to R-334; M-1 to Q-333; M-1 to Q-332; M-1 to S-331; M-1 toG-330; M-1 to L-329; M-1 to R-328; M-1 to N-327; M-1 to E-326; M-1 toA-325; M-1 to R-324; M-1 to C-323; M-1 to T-322; M-1 to Y-321; M-1 toR-320; M-1 to G-319; M-1 to S-318; M-1 to D-317; M-1 to G-316; M-1 toA-315; M-1 to K-314; M-1 to V-313; M-1 to G-312; M-1 to P-311; M-1 toL-310; M-1 to E-309; M-1 to L-308; M-1 to G-307; M-1 to L-306; M-1 toP-305; M-1 to R-304; M-1 to P-303; M-1 to G-302; M-1 to W-301; M-1 toP-300; M-1 to H-299; M-1 to S-298; M-1 to S-297; M-1 to S-296; M-1 toL-295; M-1 to V-294; M-1 to R-293; M-1 to N-292; M-1 to Q-291; M-1 toL-290; M-1 to V-289; M-1 to W-288; M-1 to S-287; M-1 to L-286; M-1 toT-285; M-1 to A-284; M-1 to P-283; M-1 to P-282; M-1 to Q-281; M-1 toS-280; M-1 to D-279; M-1 to A-278; M-1 to A-277; M-1 to C-276; M-1 toL-275; M-1 to L-274; M-1 to R-273; M-1 to L-272; M-1 to F-271; M-1 toQ-270; M-1 to G-269; M-1 to K-268; M-1 to Q-267; M-1 to A-266; M-1 toE-265; M-1 to L-264; M-1 to Y-263; M-1 to P-262; M-1 to V-261; M-1 toN-260; M-1 to G-259; M-1 to Q-258; M-1 to P-257; M-1 to Q-256; M-1 toP-255; M-1 to E-254; M-1 to L-253; M-1 to A-252; M-1 to P-251; M-1 toT-250; M-1 to N-249; M-1 to D-248; M-1 to R-247; M-1 to S-246; M-1 toI-245; M-1 to S-244; M-1 to I-243; M-1 to V-242; M-1 to L-241; M-1 toD-240; M-1 to R-239; M-1 to P-238; M-1 to A-237; M-1 to Y-236; M-1 toA-235; M-1 to V-234; M-1 to R-233; M-1 to M-1 to L-232; M-1 to R-231;M-1 to V-230; M-1 to T-229; M-1 to R-228; M-1 to Q-227; M-1 to V-226;M-1 to S-225; M-1 to V-224; M-1 to G-223; M-1 to K-222; M-1 to R-221;M-1 to S-220; M-1 to F-219; M-1 to D-218; M-1 to V-217; M-1 to H-216;M-1 to M-1 to C-215; M-1 to T-214; M-1 to L-213; M-1 to D-212; M-1 toT-211; M-1 to N-210; M-1 to H-209; M-1 to D-208; M-1 to Q-207; M-1 toP-206; M-1 to R-205; M-1 to P-204; M-1 to T-203; M-1 to F-202; M-1 toS-201; M-1 to L-200; M-1 to V-199; M-1 to S-198; M-1 to F-197; M-1 toH-196; M-1 to S-195; M-1 to T-194; M-1 to T-193; M-1 to P-192; M-1 toK-191; M-1 to T-190; M-1 to G-189; M-1 to Q-188; M-1 to S-187; M-1 toS-186; M-1 to L-185; M-1 to A-184; M-1 to A-183; M-1 to G-182; M-1 toT-181; M-1 to W-180; M-1 to S-179; M-1 to F-178; M-1 to S-177; M-1 toP-176; M-1 to P-175; M-1 to P-174; M-1 to C-173; M-1 to E-172; M-1 toE-171; M-1 to F-170; M-1 to A-169; M-1 to W-168; M-1 to N-167; M-1 toF-166; M-1 to V-165; M-1 to C-164; M-1 to I-163; M-1 to V-162; M-1 toT-161; M-1 to V-160; M-1 to P-159; M-1 to Q-158; M-1 to G-157; M-1 toP-156; M-1 to E-155; M-1 to L-154; M-1 to T-153; M-1 to E-152; M-1 toP-151; M-1 to I-150; M-1 to Y-149; M-1 to V-148; M-1 to D-147; M-1 toP-146; M-1 to K-145; M-1 to Q-144; M-1 to T-143; M-1 to L-142; M-1 toA-141; M-1 to T-140; M-1 to V-139; M-1 to K-138; M-1 to L-137; M-1 toF-136; M-1 to F-135; M-1 to G-134; M-1 to D-133; M-1 to N-132; M-1 toM-131; M-1 to F-130; M-1 to N-129; M-1 to Y-128; M-1 to R-127; M-1 toV-126; M-1 to Y-125; M-1 to S-124; M-1 to G-123; M-1 to R-122; M-1 toE-121; M-1 to V-120; M-1 to R-119; M-1 to F-118; M-1 to F-117; M-1 toY-116; M-1 to Q-115; M-1 to S-114; M-1 to E-113; M-1 to D-112; M-1 toQ-111; M-1 to M-110; M-1 to Q-109; M-1 to A-108; M-1 to D-107; M-1 toR-106; M-1 to I-105; M-1 to V-104; M-1 to L-103; M-1 to S-102; M-1 toC-101; M-1 to N-100; M-1 to G-99; M-1 to K-98; M-1 to A-97; M-1 to P-96;M-1 to D-95; M-1 to G-94; M-1 to T-93; M-1 to L-92; M-1 to Q-91; M-1 toF-90; M-1 to R-89; M-1 to G-88; M-1 to R-87; M-1 to T-86; M-1 to S-85;M-1 to M-84; M-1 to E-83; M-1 to V-82; M-1 to E-81; M-1 to R-80; M-1 toS-79; M-1 to Q-78; M-1 to H-77; M-1 to N-76; M-1 to T-75; M-1 to A-74;M-1 to V-73; M-1 to P-72; M-1 to A-71; M-1 to G-70; M-1 to K-69; M-1 toT-68; M-1 to T-67; M-1 to E-66; M-1 to T-65; M-1 to V-64; M-1 to A-63;M-1 to K-62; M-1 to F-61; M-1 to W-60; M-1 to Y-59; M-1 to G-58; M-1 toY-57; M-1 to A-56; M-1 to P-55; M-1 to T-54; M-1 to S-53; M-1 to G-52;M-1 to T-51; M-1 to W-50; M-1 to D-49; M-1 to Q-48; M-1 to R-47; M-1 toP-46; M-1 to Y-45; M-1 to S-44; M-1 to F-43; M-1 to S-42; M-1 to C-41;M-1 to P-40; M-1 to V-39; M-1 to S-38; M-1 to I-37; M-1 to C-36; M-1 toL-35; M-1 to G-34; M-1 to E-33; M-1 to P-32; M-1 to V-31; M-1 to M-30;M-1 to V-29; M-1 to S-28; M-1 to E-27; M-1 to Q-26; M-1 to V-25; M-1 toR-24; M-1 to I-23; M-1 to W-22; M-1 to F-21; M-1 to R-20; M-1 to G-19;M-1 to D-18; M-1 to M-17; M-1 to A-16; M-1 to Q-15; M-1 to S-14; M-1 toG-13; M-1 to G-12; M-1 to L-11; M-1 to L-10; M-1 to S-9; M-1 to S-8; andM-1 to L-7 of SEQ ID NO: 149. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g. fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:15and/or SEQ ID NO: 148, which have been determined in part from thefollowing related cDNA genes: HTOFA26R (SEQ ID NO:93), HWAEM43R (SEQ IDNO:94), HDPMQ69R (SEQ ID NO:95), HDPGA09RA (SEQ ID NO:96), HEOMH10R (SEQID NO:97), and HFKCT73F (SEQ ID NO:98).

The CD33-like polypeptide sequence corresponding to SEQ ID NO: 33 ofthis gene has been determined to have a transmembrane domain at aboutamino acid position 496-512 of the amino acid sequence referenced inTable XIV for this gene. Moreover, a cytoplasmic tail encompassing aminoacids 513 to 639 of this protein has also been determined. Based uponthese characteristics, it is believed that the protein product of thisgene shares structural features to type Ia membrane proteins.

The CD33-likeSV polypeptide sequence corresponding to SEQ ID NO: 149 hasbeen determined to have a transmembrane domain at about amino acidposition 539-572 of SEQ ID NO: 149. Moreover, a cytoplasmic tailencompassing amino acids 573-697 of SEQ ID NO: 149 has also beendetermined. Additionally, polypeptides encompassed by the inventioncomprise, or alternatively consist of, one, two, three, four, or allfive of the CD33-likeSV Ig-like domains represented in SEQ ID NO: 149 asamino acids Phe-21 to Ala-141; Leu-142 to Tyr-236; Leu-253 to Gln-341;Val-358 to Val-442; and His-443 to Pro-538 (See FIG. 37 for domains).

Northern analysis indicates that CD33-like is expressed highest inspleen tissue and peripheral blood leukocytes, and to a lesser extent inovary and lung tissue.

Additionally, Northern blot analysis reveals the presence of a majorCD33-likeSV mRNA transcript of approximately 3.0 kb, with highest levelsin spleen, lymph node, blood leukocytes, and appendix. CD33-likeSV isalso expressed in eosinophils, B cells and monocytes.

Therefore, CD33-like polynucleotides and polypeptides, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of the following diseases and conditions which include, butare not limited to, disorders of the immune system, in particular theimmunodiagnosis of acute leukemias. Similarly, polypeptides andantibodies directed to these polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels is detected in certain tissues orcell types (e.g., immune, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid or spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue from an individual not having thedisorder.

Similarly, CD33-likeSV polynucleotides and polypeptides, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of the following diseases and conditions which include, butare not limited to, disorders of the immune system, includinginflammatory and allergic disorders. Similarly, polypeptides andantibodies directed to these polypeptides are useful to provideimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels is detected in certain tissues orcell types (e.g., immune, cancerous and wounded tissues) or bodilyfluids (e.g., lymph, serum, plasma, urine, synovial fluid or spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue from an individual not having thedisorder.

CD33 monoclonal antibodies (MoAB) are important in the immunodiagnosisof AML. CD33 MoABs have been used in preliminary therapeutic trials forpurging bone marrow of AML patients, either before transplantation orfor diseases resistant to chemotherapy. To prevent AML patients inremission from suffering relapse, or due to the lack of an appropriateallogenic bone marrow donor, a method is necessary for purging leukemiacells from the autografts of patients with advanced AML. By theinvention, this method is provided by which bone marrow from an AMLpatient is obtained by, for example, percutaneous aspirations from theposterior iliac crest, isolating bone marrow mononuclear byFicoll-hypaque density gradient centrifugation, and incubating with ananti-CD33-like protein MoAB, for example, 3-5 times for 15-30 min. at4-6 degrees C., followed by incubation with rabbit complement at about37 degrees C. for 30 minutes. The patient is then subject tomyeloablative chemotherapy, followed by reinfusion of the treatedautologous bone marrow according to standard techniques. By theinvention, clonogenic tumor cells are depleted from the bone marrowwhile sparing hematopoietic cells necessary for engraftment.

In a further embodiment,the invention provides an in vivo method forselectively killing or inhibiting growth of tumor cells expressingCD33-like protein antigen of the present invention. The method involvesadministering to the patient an effective amount of an antagonist toinhibit the CD33-like protein receptor signaling pathway. By theinvention, administering such antagonist of the CD33-like protein to apatient may also be useful for treating inflammatory diseases includingarthritis and colitis. Antagonists for use in the present inventioninclude polyclonal and monoclonal antibodies raised against theCD33-like protein or a fragment thereof, antisense molecules whichcontrol gene expression through antisense DNA or RNA or throughtriple-helix formation, proteins or other compounds which bind theCD33-like protein domains, or soluble forms of the CD33-like protein,such as protein fragments including the extracellular region from thefull length receptor, which antagonize CD33-like protein mediatedsignaling by competing with the cell surface CD33-like protein forbinding to CD33 receptor ligands.

The tissue distribution of CD33-likeSV in blood leukocytes, B cells andeosinophils, and the homology to other CD33 related Siglecs, indicatesthat polynucleotides, translation products and antibodies correspondingto CD33-like and CD33-likeSV are useful for the diagnosis, detectionand/or treatment of diseases and/or disorders of the immune system.

The homology to a number of CD33 related Siglec proteins indicates thatpolynucleotides, translation products and antibodies corresponding toCD33-like and CD33-likeSV may be useful as a target for immunotherapy indiseases and/or disorders involving the immune system, and moreparticularly diseases and/or disorders related to leukocytes. Theexpression in eosinophils indicates that polynucleotides, translationproducts and antibodies corresponding to CD33-like and/or CD33-likeSVare useful for the diagnosis, detection and/or treatment of diseasesand/or disorders related to eosinophils, such as, for example,eosinophil adenoma, eosinophilic granuloma, eosinophilic leukemia, andeosinophilic nonallergic rhinitis, allergic disorders, and/orhypereosinophilic disorders. Additionally, the expression in bloodleukocytes indicates that polynucleotides, translation products andantibodies corresponding to CD33-like and CD33-likeSV are useful for thediagnosis, detection and/or treatment of diseases and/or disordersrelating to other blood leukocytes, such as monocytes, for example.

Alternatively, the homology to other CD33 related Siglec proteinsindicates that translation products corresponding to CD33-like andCD33-likeSV may play a useful role in the activation of cells involvedin the destruction of pathogens. This may occur, by way of anon-limiting hypothesis, through an interaction between CD33-like and/orCD33-likeSV translation products and non-sialylated pathogens.Furthermore, the binding of CD33-like and/or CD33-likeSV translationproducts to erythrocytes suggests that these translation products mayfunction in the elimination of erythrocytes from the blood stream and/orself-recognition, and may play a role in the recognition of sialic acidon the surface of erythrocytes. Sialidase treated erythrocytes were notbound by translation products corresponding to CD33-like and/orCD33-likeSV. Thus, translation products corresponding to CD33-likeand/or CD33-likeSV may be involved in the clearance of erythrocytes fromthe blood stream, self-recognition and/or immune system activation.

Polynucleotides or polypeptides, or agonists or antagonists of thepresent invention, can be used in assays to test for one or morebiological activities. If these polynucleotides or polypeptides, oragonists or antagonists of the present invention, do exhibit activity ina particular assay, it is likely that these molecules may be involved inthe diseases associated with the biological activity. Thus, thepolynucleotides and polypeptides, and agonists or antagonists could beused to treat the associated disease.

CD33-like and CD33-likeSV polypeptides are believed to be involved inbiological activities associated with immune cell activation, pathogenrecognition, erythrocyte binding, and/or eosinophilic and other immunecell disorders. Accordingly, compositions of the invention (includingpolynucleotides, polypeptides and antibodies of the invention, andfragments and variants thereof) may be used in the diagnosis, detectionand/or treatment of diseases and/or disorders associated with aberrantCD33-like and CD33-likeSV activity. In preferred embodiments,compositions of the invention (including polynucleotides, polypeptidesand antibodies of the invention, and fragments and variants thereof) maybe used in the diagnosis, detection and/or treatment of diseases and/ordisorders relating to blood disorders (e.g., polycythemia, and/or asdescribed under “Immune activity” and “Cardiovascular Disorders” below),hypereosinophilic disorders, allergic disorders, pathogen recognition,erythrocyte binding, and immune system disorders (e.g., pathogeninfections, and/or as described under “Immune activity” below). Thus,polynucleotides, translation products and antibodies of the inventionare useful in the diagnosis, detection and/or treatment of diseasesand/or disorders associated with activities that include, but are notlimited to, blood disorders, hypereosinophilic disorders, allergicdisorders, pathogen recognition, erythrocyte binding, and immune systemdisorders.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO: 15 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2281 of SEQID NO:15, b is an integer of 15 to 2295, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:15, and whereb is greater than or equal to a+14.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:148 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence would be cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 2324 of SEQID NO:148, b is an integer of 15 to 2338, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:148, andwhere b is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 6

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. The polypeptide of the present invention has beenputatively identified as a CD33 homolog derived from a human primarydendritic cells cDNA library. More particularly, the polypeptide of thepresent invention has been putatively identified as a human siglechomolog, sometimes hereafter referred to as “CD33-like 3” and/or “siglec7”. The invention also relates to inhibiting the action of suchpolypeptides.

The siglecs (sialic acid binding Ig-like lectins) are type I membraneproteins that constitute a distinct subset of the Ig superfamily,characterised by their sequence similarities and abilities to bindsialic acids in glycoproteins and glycolipid (Crocker, P. R., et al.,Glycobiology: 8 (1998)). Members of the Ig Superfamily of proteins aredefined as molecules that share domains of sequence similarity with thevariable or constant domains of antibodies.

Many Ig superfamily proteins consist of multiple tandem Ig-like domainsconnected to other domains, such as Fn-III repeat domains (Vaughn, D.E., and P. J. Bjorkman, Neuron, 16:261-73 (1996)). At the primarystructural level, traditional Ig-like domains can be identified by thepresence of two cysteine residues separated by approximately 55-75 aminoacid residues, and an “invariant”tryptophan residue located 10-15residues C-terminal to the first of the two conserved cysteine residues.The two conserved cysteine residues are thought to be involved indisulfide bonding to form the folded Ig structures (Vaughn, D. E.,(1996)).

Ig-like domains further share a common folding pattern, that of asandwich or fold structure of two b-sheets consisting of antiparallelb-strands containing 5-10 amino acids (Huang, Z., et al., Biopolymers,43:367-82 (1997)). Ig-like domains are divided, based upon sequence andstructural similarities, into four classifications known as C1, C2, Iand V-like domains.

The functional determinants of the Ig-like domains are presented on thefaces of b-sheets or the loop regions of the Ig-fold. Accordingly,protein-protein interactions can occur either between the faces of theb-sheets, or the loop regions of the Ig-fold (Huang, Z., (1998)). TheseIg-like domains are involved in mediating a diversity of biologicalfunctions such as intermolecular binding and protein-protein homophilicor heterophilic interactions.

Thus, Ig-like domains play an integral role in facilitating theactivities of proteins of the Ig superfamily. In mammals, the groupcurrently comprises sialoadhesin/siglec-1, CD22/siglec-2, CD33/siglec-3,myelin associated glycoprotein (MAG/siglec-4), siglecs-5, -6 and -7(Crocker, P. R., et al., EMBO J., 13:4490-503 (1994); Sgroi, D., et al.,J Biol. Chem. 268:7011-18 (1993); Freeman, D. S., et al., Blood,85:2005-12 (1995); Kelm, S., et al., Curr Biol., 4:965-72 (1994);Cornish, A. L., et al., Blood, 92:2123-132 (1998); Patel, N., et al., JBiol. Chem, 274:22729-738 (1999); Nicol, G., et al., J Biol. Chem, InPress (1999)). Siglec-7 has also recently been characterisedindependently as the NK receptor p75/AIRM1 (Falco, M., et al., J Exp.Med., 190:793-802 (1999)). In addition, the gene encoding anothersiglec-like sequence, OBBP-like protein has been reported but there isno information on its binding activity (Yousef, G. M., et al., Biochem.Biophys. Res. Commun., In Press (1999)).

Each of these proteins has an extracellular region made up of a membranedistal V-set domain followed by varying numbers of C2 set domains whichrange from 16 in sialoadhesin to 1 in CD33. In the cases ofsialoadhesin, CD22, MAG and CD33, the sialic acid binding site has beenmapped to the V-set domain and for sialoadhesin it has been furthercharacterised at the molecular level by X-ray crystallography 11 (Nath,D., et al., J Biol. Chem., 270:26184-91 (1995); van der Merwe, P. A., etal., J. Biol. Chem., 271:9273-80 (1996); Tang, S., et al., J. CellBiol., 138:1355-66 (1997); Taylor, V. C., et al., J. Biol. Chem.,274:11505-12 (1999); May, A. P., et al., Molecular Cell, 1:719-28(1998)).

Apart from MAG and SMP that are found exclusively in the nervous system,all siglecs characterised to date are expressed on discrete subsets ofhemopoietic cells and can provide useful lineage-restricted markers.Thus, CD22 is present only on mature B cells, sialoadhesin is onmacrophage subsets, CD33 is a marker of early committed myeloidprogenitor cells, siglec-5 is expressed by monocytes and matureneutrophils, siglec-6 is on B cells and siglec-7 is expressed by NKcells and monocytes (Dorken, B., et al., J. Immunology, 136:4470-79(1986); Crocker, P. R., et al., J. Exp. Med., 164:1862-75 (1986);Peiper, S. C., et al., In Leukocyte Typing IV. Oxford University Press,Oxford. 814-16 (1989); Cornish, A. L., et al., Blood, 92:2123-132(1998); Patel, N., et al., J Biol. Chem, 274:22729-738 (1999); Nicol,G., et al., J Biol. Chem, In Press (1999)).

These expression patterns indicate discrete functions amongsthemopoietic cell subsets, but apart from CD22, a well-characterisednegative regulator of B cell activation (reviewed in Cyster, J. G. andC. C. Goodnow, Immunity, 6:509-17 (1997)), the biological functions ofsiglecs expressed in the hemopoietic system are unknown. Proposedfunctions include cell-cell interactions through recognition ofsialylated glycoconjugates on other cells. However, a number of studieshave also shown that cell-cell adhesion mediated by siglecs can bemodulated by cis-interactions with sialic acids present in the hostplasma membrane. This is particularly striking for CD22, CD33 andsiglec-5, whose binding activities can be greatly increased if hostcells are pretreated with sialidase to remove the cis-competing sialicacids (Freeman, D. S., et al., Blood, 85:2005-12 (1995); Cornish, A. L.,et al., Blood, 92:2123-132 (1998); Sgroi, D., et al., P.N.A.S.,92:4026-30 (1995)).

Besides potential roles in cellular interactions, there is growingevidence that, similar to CD22, the more recently characterised siglecsare involved in signalling functions. The cytoplasmic tails of CD33 andsiglecs-5, -6 and -7 have two well-conserved tyrosine-based motifs thatare similar to well-characterised signaling motifs in other leukocytereceptors (Gergely, J., et al., Immun. Lett., 68:3-15 (1999)). Wherestudied, both tyrosine residues can be phosphorylated by src-likekinase(s) and, in the case of the membrane proximal tyrosine, this leadsto subsequent recruitment of the tyrosine phosphatases, SHP-1 and SHP-2(Falco, M., et al., J. Exp. Med., 190:793-802 (1999); Taylor, V. C., etal., J. Biol. Chem., 274:11505-12 (1999)).

Thus there exists a clear need for identifying and exploiting novelmembers of the siglec family of immunoglobulin proteins. Althoughstructurally related, such proteins may possess diverse and multifacetedfunctions in a variety of cell and tissue types. The purified siglecproteins of the invention are research tools useful for theidentification, characterization and purification of cell signalingmolecules. Furthermore, the identification of new siglecs permits thedevelopment of a range of derivatives, agonists and antagonists at thenucleic acid and protein levels which in turn have applications in thetreatment and diagnosis of a range of conditions such as cancer,inflammation, neurological disorders and immunological disorders,amongst many other conditions. The polypeptide of the present inventionhas been putatively identified as a member of the siglec family and hasbeen termed CD33-like 3. This identification has been made as a resultof amino acid sequence homology to the human cd3311 (See GenbankAccession No. gi|2913995).

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, or all twelve of the immunogenicepitopes shown in SEQ ID NO: 34 as residues: Gly-12 to Tyr-26, Val-52 toAsp-59, Gln-88 to Asp-93, Arg-124 to Asn-129, His-193 to Arg-198,Gln-207 to Thr-213, Gln-338 to Arg-346, Ser-378 to Ala-384, Ser-413 toArg-420, Ser-428 to Glu-434, His-443 to Ser-451, and/or Glu-454 toSer-461. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

FIGS. 16A-B show the nucleotide (SEQ ID NO:16) and deduced amino acidsequence (SEQ ID NO:34) of CD33-like 3. Predicted amino acids from about1 to about 18 constitute the predicted signal peptide (amino acidresidues from about 1 to about 18 in SEQ ID NO:34) and are representedby the underlined amino acid regions; and amino acids from about 360 toabout 376 constitute the predicted transmembrane domain (amino acidsfrom about 360 to about 376 in SEQ ID NO:34) and are represented by thedouble underlined amino acids.

FIG. 17 shows the regions of similarity between the amino acid sequencesof the CD33-like 3 protein (SEQ ID NO:34) and the human CD33L1 protein(SEQ ID NO:99).

FIG. 18 shows an analysis of the CD33-like 3 amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

As shown in FIGS. 16A-B, CD33-like 3 has a transmembrane domain (thetransmembrane domains comprise amino acids from about 360 to about 376of SEQ ID NO:34; which correspond to amino acids from about 360 to about376 of FIGS. 16A-B). The polynucleotide contains an open reading frameencoding the CD33-like 3 polypeptide of 467 amino acids. CD33-like 3exhibits a high degree of homology at the amino acid level to the humanCD33L1 (as shown in FIG. 18).

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the CD33-like 3 polypeptide havingthe amino acid sequence shown in FIGS. 16A-B (SEQ ID NO:34). Thenucleotide sequence shown in FIGS. 16A-B (SEQ ID NO:16) was obtained bysequencing a cloned cDNA (HDPUW68), which was deposited on November 17at the American Type Culture Collection, and given Accession Number203484.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO:16 is intended DNA fragments atleast about 15nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:16. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:16. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Representative examples of CD33-like 3 polynucleotide fragmentsof the invention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, from about 501 toabout 550, from about 551 to about 600, from about 601 to about 650,from about 651 to about 700, from about 701 to about 750, from about 751to about 800, from about 801 to about 850, from about 851 to about 900,from about 901 to about 950, from about 951 to about 1000, from about1001 to about 1050, from about 1051 to about 1100, from about 1101 toabout 1150, from about 1151 to about 1200, from about 1201 to about1250, from about 1251 to about 1300, from about 1301 to about 1350, fromabout 1351 to about 1400, from about 1401 to about 1450, from about 1451to about 1500, from about 1501 to about 1550, from about 1551 to about1600, from about 1601 to about 1650, from about 1651 to about 1700, fromabout 1701 to about 1748 of SEQ ID NO:16, or the complementary strandthereto, or the cDNA contained in the deposited gene. In this context“about” includes the particularly recited ranges, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding a member selected from the group: apolypeptide comprising or alternatively, consisting of, thetransmembrane domain (amino acid residues from about 360 to about 376 inFIGS. 16A-B (amino acids from about 360 to about 376 in SEQ ID NO:34).Since the location of these domains have been predicted by computeranalysis, one of ordinary skill would appreciate that the amino acidresidues constituting these domains may vary slightly (e.g., by about 1to 15 amino acid residues) depending on the criteria used to define eachdomain.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of CD33-like 3.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of CD33-like 3. The data representing the structural orfunctional attributes of CD33-like 3 set forth in FIG. 18 and/or TableVI, as described above, was generated using the various modules andalgorithms of the DNA*STAR set on default parameters. In a preferredembodiment, the data presented in columns VIII, IX, XIII, and XIV ofTable VI can be used to determine regions of CD33-like 3 which exhibit ahigh degree of potential for antigenicity. Regions of high antigenicityare determined from the data presented in columns VIII, IX, XIII, and/orXIV by choosing values which represent regions of the polypeptide whichare likely to be exposed on the surface of the polypeptide in anenvironment in which antigen recognition may occur in the process ofinitiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 18, butmay, as shown in Table VI, be represented or identified by using tabularrepresentations of the data presented in FIG. 18. The DNA*STAR computeralgorithm used to generate FIG. 18 (set on the original defaultparameters) was used to present the data in FIG. 18 in a tabular format(See Table VI). The tabular format of the data in FIG. 18 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 18 and in Table VIinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS.16A-B. As set out in FIG. 18 and in Table VI, such preferred regionsinclude Garnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolitle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, modulate cellularinteraction, or signalling pathways, etc.) may still be retained. Forexample, the ability of shortened CD33-like 3 muteins to induce and/orbind to antibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that an CD33-like 3 mutein with alarge number of deleted N-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six CD33-like 3 amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the CD33-like 3amino acid sequence shown in FIGS. 16A-B, up to the glutamic acidresidue at position number 462 and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues n1-467 of FIGS. 16A-B,where n1 is an integer from 2 to 462 corresponding to the position ofthe amino acid residue in FIGS. 16A-B (which is identical to thesequence shown as SEQ ID NO:34). In another embodiment, N-terminaldeletions of the CD33-like 3 polypeptide can be described by the generalformula n2-467, where n2 is a number from 2 to 462, corresponding to theposition of amino acid identified in FIGS. 16A-B . N-terminal deletionsof the CD33-like 3 polypeptide of the invention shown as SEQ ID NO:34include polypeptides comprising the amino acid sequence of residues:N-terminal deletions of the CD33-like 3 polypeptide of the inventionshown as SEQ ID NO:34 include polypeptides comprising the amino acidsequence of residues: L-2 to K-467; L-3 to K-467; L-4 to K-467; L-5 toK-467; L-6 to K-467; L-7 to K-467; P-8 to K-467; L-9 to K-467; L-10 toK-467; W-11 to K-467; G-12 to K-467; R-13 to K-467; E-14 to K-467; R-15to K-467; V-16 to K-467; E-17 to K-467; G-18 to K-467; Q-19 to K-467;K-20 to K-467; S-21 to K-467; N-22 to K-467; R-23 to K-467; K-24 toK-467; D-25 to K-467; Y-26 to K-467; S-27 to K-467; L-28 to K-467; T-29to K-467; M-30 to K-467; Q-31 to K-467; S-32 to K-467; S-33 to K-467;V-34 to K-467; T-35 to K-467; V-36 to K-467; Q-37 to K-467; E-38 toK-467; G-39 to K-467; M-40 to K-467; C-41 to K-467; V-42 to K-467; H-43to K-467; V-44 to K-467; R-45 to K-467; C-46 to K-467; S-47 to K-467;F-48 to K-467; S-49 to K-467; Y-50 to K-467; P-51 to K-467; V-52 toK-467; D-53 to K-467; S-54 to K-467; Q-55 to K-467; T-56 to K-467; D-57to K-467; S-58 to K-467; D-59 to K-467; P-60 to K-467; V-61 to K-467;H-62 to K-467; G-63 to K-467; Y-64 to K-467; W-65 to K-467; F-66 toK-467; R-67 to K-467; A-68 to K-467; G-69 to K-467; N-70 to K-467; D-71to K-467; I-72 to K-467; S-73 to K-467; W-74 to K-467; K-75 to K-467;A-76 to K-467; P-77 to K-467; V-78 to K-467; A-79 to K-467; T-80 toK-467; N-81 to K-467; N-82 to K-467; P-83 to K-467; A-84 to K-467; W-85to K-467; A-86 to K-467; V-87 to K-467; Q-88 to K-467; E-89 to K-467;E-90 to K-467; T-91 to K-467; R-92 to K-467; D-93 to K-467; R-94 toK-467; F-95 to K-467; H-96 to K-467; L-97 to K-467; L-98 to K-467; G-99to K-467; D-100 to K-467; P-101 to K-467; Q-102 to K-467; T-103 toK-467; K-104 to K-467; N-105 to K-467; C-106 to K-467; T-107 to K-467;L-108 to K-467; S-109 to K-467; I-110 to K-467; R-111 to K-467; D-112 toK-467; A-113 to K-467; R-114 to K-467; M-115 to K-467; S-116 to K-467;D-117 to K-467; A-118 to K-467; G-119 to K-467; R-120 to K-467; Y-121 toK-467; F-122 to K-467; F-123 to K-467; R-124 to K-467; M-125 to K-467;E-126 to K-467; K-127 to K-467; G-128 to K-467; N-129 to K-467; I-130 toK-467; K-131 to K-467; W-132 to K-467; N-133 to K-467; Y-134 to K-467;K-135 to K-467; Y-136 to K-467; D-137 to K-467; Q-138 to K-467; L-139 toK-467; S-140 to K-467; V-141 to K-467; N-142 to K-467; V-143 to K-467;T-144 to K-467; A-145 to K-467; L-146 to K-467; T-147 to K-467; H-148 toK-467; R-149 to K-467; P-150 to K-467; N-151 to K-467; I-152 to K-467;L-153 to K-467; I-154 to K-467; P-155 to K-467; G-156 to K-467; T-157 toK-467; L-158 to K-467; E-159 to K-467; S-160 to K-467; G-161 to K-467;C-162 to K-467; F-163 to K-467; Q-164 to K-467; N-165 to K-467; L-166 toK-467; T-167 to K-467; C-168 to K-467; S-169 to K-467; V-170 to K-467;P-171 to K-467; W-172 to K-467; A-173 to K-467; C-174 to K-467; E-175 toK-467; Q-176 to K-467; G-177 to K-467; T-178 to K-467; P-179 to K-467;P-180 to K-467; M-181 to K-467; I-182 to K-467; S-183 to K-467; W-184 toK-467; M-185 to K-467; G-186 to K-467; T-187 to K-467; S-188 to K-467;V-189 to K-467; S-190 to K-467; P-191 to K-467; L-192 to K-467; H-193 toK-467; P-194 to K-467; S-195 to K-467; T-196 to K-467; T-197 to K-467;R-198 to K-467; S-199 to K-467; S-200 to K-467; V-201 to K-467; L-202 toK-467; T-203 to K-467; L-204 to K-467; I-205 to K-467; P-206 to K-467;Q-207 to K-467; P-208 to K-467; Q-209 to K-467; H-210 to K-467; H-211 toK-467; G-212 to K-467; T-213 to K-467; S-214 to K-467; L-215 to K-467;T-216 to K-467; C-217 to K-467; Q-218 to K-467; V-219 to K-467; T-220 toK-467; L-221 to K-467; P-222 to K-467; G-223 to K-467; A-224 to K-467;G-225 to K-467; V-226 to K-467; T-227 to K-467; T-228 to K-467; N-229 toK-467; R-230 to K-467; T-231 to K-467; I-232 to K-467; Q-233 to K-467;L-234 to K-467; N-235 to K-467; V-236 to K-467; S-237 to K-467; Y-238 toK-467; P-239 to K-467; P-240 to K-467; Q-241 to K-467; N-242 to K-467;L-243 to K-467; T-244 to K-467; V-245 to K-467; T-246 to K-467; V-247 toK-467; F-248 to K-467; Q-249 to K-467; G-250 to K-467; E-251 to K-467;G-252 to K-467; T-253 to K-467; A-254 to K-467; S-255 to K-467; T-256 toK-467; A-257 to K-467; L-258 to K-467; G-259 to K-467; N-260 to K-467;S-261 to K-467; S-262 to K-467; S-263 to K-467; L-264 to K-467; S-265 toK-467; V-266 to K-467; L-267 to K-467; E-268 to K-467; G-269 to K-467;Q-270 to K-467; S-271 to K-467; L-272 to K-467; R-273 to K-467; L-274 toK-467; V-275 to K-467; C-276 to K-467; A-277 to K-467; V-278 to K-467;D-279 to K-467; S-280 to K-467; N-281 to K-467; P-282 to K-467; P-283 toK-467; A-284 to K-467; R-285 to K-467; L-286 to K-467; S-287 to K-467;W-288 to K-467; T-289 to K-467; W-290 to K-467; R-291 to K-467; S-292 toK-467; L-293 to K-467; T-294 to K-467; L-295 to K-467; Y-296 to K-467;P-297 to K-467; S-298 to K-467; Q-299 to K-467; P-300 to K-467; S-301 toK-467; N-302 to K-467; P-303 to K-467; L-304 to K-467; V-305 to K-467;L-306 to K-467; E-307 to K-467; L-308 to K-467; Q-309 to K-467; V-3 10to K-467; H-311 to K-467; L-312 to K-467; G-313 to K-467; D-314 toK-467; E-315 to K-467; G-316 to K-467; E-317 to K-467; F-318 to K-467;T-319 to K-467; C-320 to K-467; R-321 to K-467; A-322 to K-467; Q-323 toK-467; N-324 to K-467; S-325 to K-467; L-326 to K-467; G-327 to K-467;S-328 to K-467; Q-329 to K-467; H-330 to K-467; V-331 to K-467; S-332 toK-467; L-333 to K-467; N-334 to K-467; L-335 to K-467; S-336 to K-467;L-337 to K-467; Q-338 to K-467; Q-339 to K-467; E-340 to K-467; Y-341 toK-467; T-342 to K-467; G-343 to K-467; K-344 to K-467; M-345 to K-467;R-346 to K-467; P-347 to K-467; V-348 to K-467; S-349 to K-467; G-350 toK-467; V-351 to K-467; L-352 to K-467; L-353 to K-467; G-354 to K-467;A-355 to K-467; V-356 to K-467; G-357 to K-467; G-358 to K-467; A-359 toK-467; G-360 to K-467; A-361 to K-467; T-362 to K-467; A-363 to K-467;L-364 to K-467; V-365 to K-467; F-366 to K-467; L-367 to K-467; S-368 toK-467; F-369 to K-467; C-370 to K-467; V-371 to K-467; I-372 to K-467;F-373 to K-467; I-374 to K-467; V-375 to K-467; V-376 to K-467; R-377 toK-467; S-378 to K-467; C-379 to K-467; R-380 to K-467; K-381 to K-467;K-382 to K-467; S-383 to K-467; A-384 to K-467; R-385 to K-467; P-386 toK-467; A-387 to K-467; A-388 to K-467; D-389 to K-467; V-390 to K-467;G-391 to K-467; D-392 to K-467; I-393 to K-467; G-394 to K-467; M-395 toK-467; K-396 to K-467; D-397 to K-467; A-398 to K-467; N-399 to K-467;T-400 to K-467; I-401 to K-467; R-402 to K-467; G-403 to K-467; S-404 toK-467; A-405 to K-467; S-406 to K-467; Q-407 to K-467; G-408 to K-467;N-409 to K-467; L-410 to K-467; T-411 to K-467; E-412 to K-467; S-413 toK-467; W-414 to K-467; A-415 to K-467; D-416 to K-467; D-417 to K-467;N-418 to K-467; P-419 to K-467; R-420 to K-467; H-421 to K-467; H-422 toK-467; G-423to K-467; L-424 to K-467; A-425 to K-467; A-426 to K-467;H-427 to K-467; S-428 to K-467; S-429 to K-467; G-430 to K-467; E-431 toK-467; E-432 to K-467; R-433 to K-467; E-434 to K-467; I-435 to K-467;Q-436 to K-467; Y-437 to K-467; A-438 to K-467; P-439 to K-467; L-440 toK-467; S-441 to K-467; F-442 to K-467; H-443 to K-467; K-444 to K-467;G-445 to K-467; E-446 to K-467; P-447 to K-467; Q-448 to K-467; D-449 toK-467; L-450 to K-467; S-451 to K-467; G-452 to K-467; Q-453 to K-467;E-454 to K-467; A-455 to K-467; T-456 to K-467; N-457 to K-467; N-458 toK-467; E-459 to K-467; Y-460 to K-467; S-461 to K-467; E-462 to K-467;of SEQ ID NO:34. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activitiesmay still be retained. For example the ability of the shortenedCD33-like 3 mutein to induce and/or bind to antibodies which recognizethe complete or mature forms of the polypeptide generally will beretained when less than the majority of the residues of the complete ormature polypeptide are removed from the C-terminus. Whether a particularpolypeptide lacking C-terminal residues of a complete polypeptideretains such immunologic activities can readily be determined by routinemethods described herein and otherwise known in the art. It is notunlikely that a CD33-like 3 mutein with a large number of deletedC-terminal amino acid residues may retain some biological or immunogenicactivities. In fact, peptides composed of as few as six CD33-like 3amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the CD33-like 3 polypeptide shown in FIGS. 16A-B, up to theleucine residue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m1 of FIG. 1, where m1is an integer from 6 to 467 corresponding to the position of the aminoacid residue in FIGS. 16A-B.

Moreover, the invention provides polynucleotides encoding polypeptidescomprising, or alternatively consisting of, the amino acid sequence ofC-terminal deletions of the CD33-like 3 polypeptide of the inventionshown as SEQ ID NO:34 include polypeptides comprising the amino acidsequence of residues: M-1 to P-466; M-1 to I-465; M-1 to K-464; M-1 toI-463; M-1 to E-462; M-1 to S-461; M-1 to Y-460; M-1 to E-459; M-1 toN-458; M-1 to N-457; M-1 to T-456; M-1 to A-455; M-1 to E-454; M-1 toQ-453; M-1 to G-452; M-1 to S-451; M-1 to L-450; M-1 to D-449; M-1 toQ-448; M-1 to P-477; M-1 to E-446; M-1 to G-445; M-1 to K-444; M-1 toH-443; M-1 to F-442; M-1 to S-441; M-1 to L-440; M-1 to P-339; M-1 toA-438; M-1 to Y-437; M-1 to Q-436; M-1 to I-435; M-1 to E-434; M-1 toR-433; M-1 to E-432; M-1 to E-431; M-1 to G-430; M-1 to S-429; M-1 toS-428; M-1 to H-427; M-1 to A-426; M-1 to A-425; M-1 to L-424; M-1 toG-423; M-1 to H-422; M-1 to H-421; M-1 to R-420; M-1 to P-419; M-1 toN-418; M-1 to D-417; M-1 to D-416; M-1 to A-415; M-1 to W-414; M-1 toS-413; M-1 to E-412; M-1 to T-411; M-1 to L-410; M-1 to N-409; M-1 toG-408; M-1 to Q-407; M-1 to S-406; M-1 to A-405; M-1 to S-404; M-1 toG-403; M-1 to R-402; M-1 to I-401; M-1 to T-400; M-1 to N-399; M-1 toA-398; M-1 to D-397; M-1 to K-396; M-1 to M-395; M-1 to G-394; M-1 toI-393; M-1 to D-392; M-1 to G-391; M-1 to V-390; M-1 to D-389; M-1 toA-388; M-1 to A-387; M-1 to P-386; M-1 to R-385; M-1 to A-384; M-1 toS-383; M-1 to K-382; M-1 to K-381; M-1 to R-380; M-1 to C-379; M-1 toS-378; M-1 to R-377; M-1 to V-376; M-1 to V-375; M-1 to I-374; M-1 toF-373; M-1 to I-372; M-1 to V-371; M-1 to C-370; M-1 to F-369; M-1 toS-368; M-1 to L-367; M-1 to F-366; M-1 to V-365; M-1 to L-364; M-1 toA-363; M-1 to T-362; M-1 to A-361; M-1 to G-360; M-1 to A-359; M-1 toG-358; M-1 to G-357; M-1 to V-356; M-1 to A-355; M-1 to G-354; M-1 toL-353; M-1 to L-352; M-1 to V-351; M-1 to G-350; M-1 to S-349; M-1 toV-348; M-1 to P-347; M-1 to R-346; M-1 to M-345; M-1 to K-344; M-1 toG-343; M-1 to T-342; M-1 to Y-341; M-1 to E-340; M-1 to Q-339; M-1 toQ-338; M-1 to L-337; M-1 to S-336; M-1 to L-335; M-1 to N-334; M-1 toL-333; M-1 to S-332; M-1 to V-331; M-1 to H-330; M-1 to Q-329; M-1 toS-328; M-1 to G-327; M-1 to L-326; M-1 to S-325; M-1 to N-324; M-1 toQ-323; M-1 to A-322; M-1 to R-321; M-1 to C-320; M-1 to T-319; M-1 toF-318; M-1 to E-317; M-1 to G-316; M-1 to E-315; M-1 to D-314; M-1 toG-313; M-1 to L-312; M-1 to H-311; M-1 to V-310; M-1 to Q-309; M-1 toL-308; M-1 to E-307; M-1 to L-306; M-1 to V-305; M-1 to L-304; M-1 toP-303; M-1 to N-302; M-1 to S-301; M-1 to P-300; M-1 to Q-299; M-1 toS-298; M-1 to P-297; M-1 to Y-296; M-1 to L-295; M-1 to T-294; M-1 toL-293; M-1 to S-292; M-1 to R-291; M-1 to W-290; M-1 to T-289; M-1 toW-288; M-1 to S-287; M-1 to L-286; M-1 to R-285; M-1 to A-284; M-1 toP-283; M-1 to P-282; M-1 to N-281; M-1 to S-280; M-1 to D-279; M-1 toV-278; M-1 to A-277; M-1 to C-276; M-1 to V-275; M-1 to L-274; M-1 toR-273; M-1 to L-272; M-1 to S-271; M-1 to Q-270; M-1 to G-269; M-1 toE-268; M-1 to L-267; M-1 to V-266; M-1 to S-265; M-1 to L-264; M-1 toS-263; M-1 to S-262; M-1 to S-261; M-1 to N-260; M-1 to G-259; M-1 toL-258; M-1 to A-257; M-1 to T-256; M-1 to S-255; M-1 to A-254; M-1 toT-253; M-1 to G-252; M-1 to E-251; M-1 to G-250; M-1 to Q-249; M-1 toF-248; M-1 to V-247; M-1 to T-246; M-1 to V-245; M-1 to T-244; M-1 toL-243; M-1 to N-242; M-1 to Q-241; M-1 to P-240; M-1 to P-239; M-1 toY-238; M-1 to S-237; M-1 to V-236; M-1 to N-235; M-1 to L-234; M-1 toQ-233; M-1 to I-232; M-1 to T-231; M-1 to R-230; M-1 to N-229; M-1 toT-228; M-1 to T-227; M-1 to V-226; M-1 to G-225; M-1 to A-224; M-1 toG-223; M-1 to P-222; M-1 to L-221; M-1 to T-220; M-1 to V-219; M-1 toQ-218; M-1 to C-217; M-1 to T-216; M-1 to L-215; M-1 to S-214; M-1 toT-213; M-1 to G-212; M-1 to H-211; M-1 to H-210; M-1 to Q-209; M-1 toP-208; M-1 to Q-207; M-1 to P-206; M-1 to I-205; M-1 to L-204; M-1 toT-203; M-1 to L-202; M-1 to V-201; M-1 to S-200; M-1 to S-199; M-1 toR-198; M-1 to T-197; M-1 to T-196; M-1 to S-195; M-1 to P-194; M-1 toH-193; M-1 to L-192; M-1 to P-191; M-1 to S-190; M-1 to V-189; M-1 toS-188; M-1 to T-187; M-1 to G-186; M-1 to M-185; M-1 to W-184; M-1 toS-183; M-1 to I-182; M-1 to M-181; M-1 to P-180; M-1 to P-179; M-1 toT-178; M-1 to G-177; M-1 to Q-176; M-1 to E-175; M-1 to C-174; M-1 toA-173; M-1 to W-172; M-1 to P-171; M-1 to V-170; M-1 to S-169; M-1 toC-168; M-1 to T-167; M-1 to L-166; M-1 to N-165; M-1 to Q-164; M-1 toF-163; M-1 to C-162; M-1 to G-161; M-1 to S-160; M-1 to E-159; M-1 toL-158; M-1 to T-157; M-1 to G-156; M-1 to P-155; M-1 to I-154; M-1 toL-153; M-1 to I-152; M-1 to N-151; M-1 to P-150; M-1 to R-149; M-1 toH-148; M-1 to T-147; M-1 to L-146; M-1 to A-145; M-1 to T-144; M-1 toV-143; M-1 to N-142; M-1 to V-141; M-1 to S-140; M-1 to L-139; M-1 toQ-138; M-1 to D-137; M-1 to Y-136; M-1 to K-135; M-1 to Y-134; M-1 toN-133; M-1 to W-132; M-1 to K-131; M-1 to I-130; M-1 to N-129; M-1 toG-128; M-1 to K-127; M-1 to E-126; M-1 to M-125; M-1 to R-124; M-1 toF-123; M-1 to F-122; M-1 to Y-121; M-1 to R-120; M-1 to G-119; M-1 toA-118; M-1 to D-117; M-1 to S-116; M-1 to M-115; M-1 to R-114; M-1 toA-113; M-1 to D-112; M-1 to R-111; M-1 to I-110; M-1 to S-109; M-1 toL-108; M-1 to T-107; M-1 to C-106; M-1 to N-105; M-1 to K-104; M-1 toT-103; M-1 to Q-102; M-1 to P-101; M-1 to D-100; M-1 to G-99; M-1 toL-98; M-1 to L-97; M-1 to H-96; M-1 to F-95; M-1 to R-94; M-1 to D-93;M-1 to R-92; M-1 to T-91; M-1 to E-90; M-1 to E-89; M-1 to Q-88; M-1 toV-87; M-1 to A-86; M-1 to W-85; M-1 to A-84; M-1 to P-83; M-1 to N-82;M-1 to N-81; M-1 to T-80; M-1 to A-79; M-1 to V-78; M-1 to P-77; M-1 toA-76; M-1 to K-75; M-1 to W-74; M-1 to S-73; M-1 to I-72; M-1 to D-71;M-1 to N-70; M-1 to G-69; M-1 to A-68; M-1 to R-67; M-1 to R-66; M-1 toW-65; M-1 to Y-64; M-1 to G-63; M-1 to H-62; M-1 to V-61; M-1 to P-60;M-1 to D-59; M-1 to S-58; M-1 to D-57; M-1 to T-56; M-1 to Q-55; M-1 toS-54; M-1 to D-53; M-1 to V-52; M-1 to P-51; M-1 to Y-50; M-1 to S-49;M-1 to F-48; M-1 to S-47; M-1 to C-46; M-1 to R-45; M-1 to V-44; M-1 toH-43; M-1 to V-42; M-1 to C-41; M-1 to M-40; M-1 to G-39; M-1 to E-38;M-1 to Q-37; M-1 to V-36; M-1 to T-35; M-1 to V-34; M-1 to S-33; M-1 toS-32; M-1 to Q-31; M-1 to M-30; M-1 to T-29; M-1 to L-28; M-1 to S-27;M-1 to Y-26; M-1 to D-25; M-1 to K-24; M-1 to R-23; M-1 to N-22; M-1 toS-21; M-1 to K-20; M-1 to Q-19; M-1 to G-18; M-1 to E-17; M-1 to V-16;M-1 to R-15; M-1 to E-14; M-1 to R-13; M-1 to G-12; M-1 to W-11; M-1 toL-10; M-1 to L-9; M-1 to P-8; M-1 to L-7; M-1 to L-6; of SEQ ID NO:34.Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules havingnrucleotide sequences related to extensive portions of SEQ ID NO:16which have been determined from the following related cDNA genes:HGBAY02R (SEQ ID NO:100) and HLYBY62R (SEQ ID NO:101).

A polynucleotide encoding a polypeptide of the present invention isobtained from human NK cells, T-cells, primary dendritic cells,placenta, spleen, primary breast cancer, gall bladder, apoptotict-cells, macrophage, and chronic lymphocytic leukemia spleen. Thepolynucleotide of this invention was discovered in a human primarydendritic cell cDNA library.

Based on the sequence similarity to the human CD33L1, translationproduct of this gene is expected to share at least some biologicalactivities with CD33 proteins, and specifically myeloid modulatoryproteins and/or siglec proteins. Such activities are known in the art,some of which are described elsewhere herein.

Specifically, polynucleotides and polypeptides of the invention,including antibodies, are also useful for modulating the differentiationof normal and malignant cells, modulating the proliferation and/ordifferentiation of cancer and neoplastic cells, and modulating theimmune response. Polynucleotides and polypeptides of the invention mayrepresent a diagnostic marker for hematopoietic and immune diseasesand/or disorders. The full-length protein should be a secreted protein,based upon homology to the CD33 family. Therefore, it is secreted intoserum, urine, or feces and thus the levels is assayable from patientsamples. Assuming specific expression levels are reflective of thepresence of immune disorders, this protein would provide a convenientdiagnostic for early detection. In addition, expression of this geneproduct may also be linked to the progression of immune diseases, andtherefore may itself actually represent a therapeutic or therapeutictarget for the treatment of cancer.

Polynucleotides and polypeptides of the invention may play an importantrole in the pathogenesis of human cancers and cellular transformation,particularly those of the immune and hematopoietic systems.Polynucleotides and polypeptides of the invention may also be involvedin the pathogenesis of developmental abnormalities based upon itspotential effects on proliferation and differentiation of cells andtissue cell types. Due to the potential proliferating anddifferentiating activity of said polynucleotides and polypeptides,polynucleotides, translation products and antibodies corresponding tothis gene are useful as a therapeutic agent(s) in inducing tissueregeneration, for treating inflammatory conditions (e.g., inflammatorybowel syndrome, diverticulitis, etc.). Moreover, the invention is usefulin modulating the immune response to aberrant polypeptides, as may existin rapidly proliferating cells and tissue cell types, particularly inadenocarcinoma cells, and other cancers.

This gene is expressed predominantly on NK cells, and to a lesser extenton T-cells. Therefore, polynucleotides, translation products andantibodies corresponding to this gene are useful as reagents fordifferential identification of the tissue(s) or cell type(s) present ina biological sample and for diagnosis of the following diseases andconditions which include, but are not limited to, immune disorders andcancer, as well as the immunodiagnosis of acute leukemias. Similarly,polynucleotides, translation products and antibodies corresponding tothis gene are useful to provide immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the immunesystem, and breast tissue, expression of this gene at significantlyhigher or lower levels is detected in certain tissues or cell types(e.g. immune, cancerous and wounded tissues) or bodily fluids (e.g.,serum, plasma, urine, synovial fluid or spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue from an individual not having the disorder.

The tissue distribution in NK cells, in combination with the homology tosiglec family of proteins, indicates that polynucleotides, translationproducts and antibodies corresponding to this gene are useful for thediagnosis and treatment of a variety of immune system disorders. NKcells are bone-marrow derived granular lymphocytes that play animportant role in natural immunity to infectious diseases and have thecapacity to kill certain virally-infected cells and tumor cells thathave down-regulated MHC Class-I antigen expression. The killing andproinflammatory activities of NK cells are regulated through a varietyof cell surface receptors that can mediate either activity or inhibitorysignals. The best understood receptors are those that recognize MHCClass I molecules at the cell surface and deliver a negative signal,thereby protecting normal host cells from cytotoxicity. These receptorscan belong either to the C-type lectin superfamily or the Igsuperfamily, although in humans the majority are members of the Igsuperfamily known as killer cell Ig-like receptors (KIRs).Representative uses are described in the “Immune Activity” and“infectious disease” sections below, in Example 11, 13, 14, 16, 18, 19,20, and 27, and elsewhere herein.

Briefly, the expression indicates a role in regulating theproliferation; survival; differentiation; and/or activation ofhematopoietic cell lineages, including blood stem cells. Involvement inthe regulation of cytokine production, antigen presentation, or otherprocesses indicates a usefulness for treatment of cancer (e.g. byboosting immune responses). Expression in cells of lymphoid origin,indicates the natural gene product is involved in immune functions.Therefore it would also be useful as an agent for immunologicaldisorders including arhritis, asthma, immunodeficiency diseases such asAIDS, leukemia, rheumatoid arthritis, granulomatous Disease,inflammatory bowel disease, sepsis, acne, neutropenia, neutrophilia,psoriasis, hypersensitivities, such as T-cell mediated cytotoxicity;immune reactions to transplanted organs and tissues, such ashost-versus-graft and graft-versus-host diseases, or autoimmunitydisorders, such as autoimmune infertility, lense tissue injury,demyelination, systemic lupus erythematosis, drug induced hemolyticanemia, rheumatoid arthritis, Sjogren's Disease, and scleroderma.Moreover, the protein may represent a secreted factor that influencesthe differentiation or behavior of other blood cells, or that recruitshematopoietic cells to sites of injury. Thus, this gene product isthought to be useful in the expansion of stem cells and committedprogenitors of various blood lineages, and in the differentiation and/orproliferation of various cell types. Based upon the tissue distributionof this protein, antagonists directed against this protein is useful inblocking the activity of this protein. Accordingly, preferred areantibodies which specifically bind a portion of the translation productof this gene.

Also provided is a kit for detecting tumors in which expression of thisprotein occurs. Such a kit comprises in one embodiment an antibodyspecific for the translation product of this gene bound to a solidsupport. Also provided is a method of detecting these tumors in anindividual which comprises a step of contacting an antibody specific forthe translation product of this gene to a bodily fluid from theindividual, preferably serum, and ascertaining whether antibody binds toan antigen found in the bodily fluid. Preferably the antibody is boundto a solid support and the bodily fluid is serum. The above embodiments,as well as other treatments and diagnostic tests (kits and methods), aremore particularly described elsewhere herein. Furthermore, the proteinmay also be used to determine biological activity, raise antibodies, astissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:16 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1734 of SEQID NO:16, b is an integer of 15 to 1748, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:16, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 7

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. The polypeptide of the present invention has beenputatively identified as a human integrin alpha 11 homolog derived froma human osteoblast II cDNA library. More particularly, the polypeptideof the present invention has been putatively identified as a humanintegrin alpha 11-subunit homolog, sometimes hereafter referred to as“integrin alpha 11”, “integrin alpha 11-subunit”, “a11”, “A11-subunit”,and/or “Integrin a11-subunit”. The invention also relates to inhibitingthe action of such polypeptides. The integrins are a large family ofcell adhesion molecules consisting of noncovalently associated abheterodimers.

We have cloned and sequenced a novel human integrin a-subunit cDNA,designated a11. The a11 cDNA encodes a protein with a 22 amino acidsignal peptide, a large 1120 residue extracellular domain that containsan I-domain of 207 residues and is linked by a transmembrane domain to ashort cytoplasmic domain of 24 amino acids. The deduced a11 proteinshows the typical structural features of integrin a-subunits and issimilar to a distinct group of a-subunits from collagen-bindingintegrins. However, it differs from most integrin a-chains by anincompletetely preserved cytoplasmic GFFKR motif.

The human ITGA11 gene was located to bands q22.3-23 on chromosome 15,and its transcripts were found predominantly in bone, cartilage as wellas in cardiac and skeletal muscle. Expression of the 5.5 kilobase a11mRNA was also detectable in ovary and small intestine.

All vertebrate cells express members of the integrin family of celladhesion molecules, which mediate cellular adhesion to other cells andextracellular subtratum, cell migration and participate in importantphysiologic processes from signal transduction to cell proliferation anddifferentiation (Hynes, 92; Springer, 92).

Integrins are structurally homologous heterodimeric type-I membraneglycoproteins formed by the noncovalent association of one of eightb-subunits with one of the 17 different a-subunits described to date,resulting in at least 22 different ab complexes. Their bindingspecificities for cellular and extracellular ligands are determined byboth subunits and are dynamically regulated in a cell-type-specific modeby the cellular environment as well as by the developmental andactivation state of the cell {Diamond and Springer, 94}. In integrina-subunits, the aminoterminal region of the large extracellular domainconsists of a seven-fold repeated structure which is predicted to foldinto a b-propeller domain {Corbi et al., 1987; Springer, 1997}. Thethree or four C-terminal repeats contain putative divalent cationbinding motifs that are thought to be important for ligand binding andsubunit association {Diamond and Springer, 94}. The a1, a2, a 10, aD,aE, aL, aM and aX-subunits contain an approximately 200 amino acidI-domain inserted between the second and third repeat that is notpresent in other a-chains {Larson et al., 1989}. Several isolatedI-domains have been shown to independently bind the ligands of theparent integrin heterodimer {Kamata and Takada, 1994; Randi and Hogg,1994}. The a3, a5-8, a11b and aV-subunits are proteolytically processedat a conserved site into disulphide-linked heavy and light chains, whilethe a4-subunit is cleaved at a more aminoterminal site into twofragments that remain noncovalently associated {Hemler et al., 90}.Additional a-subunit variants are generated by alternative splicing ofprimary transcripts {Ziober et al., 93; Delwel et al., 95; Leung et al.,98}.

The extracellular domains of a-integrin subunits are connected by asingle spanning transmembrane domain to short, diverse cytoplasmicdomains whose only conserved feature is a membrane-proximal KXGFF(K/R)Rmotif {Sastry and Horwitz, 1993}. The cytoplasmic domains have beenimplicated in the cell-type-specific modulation of integrin affinitystates {Williams et al., 1994}.

The polypeptide of the present invention has been putatively identifiedas a member of the integrin family and has been termed integrin alpha 11subunit (“a11”). This identification has been made as a result of aminoacid sequence homology to the human integrin alpha 1 subunit (SeeGenbank Accession No. gi|346210).

FIGS. 19A-F show the nucleotide (SEQ ID NO:17) and deduced amino acidsequence (SEQ ID NO:35) of a11. Predicted amino acids from about 1 toabout 22 constitute the predicted signal peptide (amino acid residuesfrom about 1 to about 22 in SEQ ID NO:35) and are represented by theunderlined amino acid regions; amino acids from about 666 to about 682,and/or amino acids from about 1145 to about 1161 constitute thepredicted transmembrane domains (amino acids from about 666 to about682, and/or amino acids from about 1145 to about 1161 in SEQ ID NO:35)and are represented by the double underlined amino acids; and aminoacids from about 64 to about 96 constitute the predicted immunoglobulinand major histocompatibility complex protein domain (amino acids fromabout 64 to about 96 in SEQ ID NO:35) and are represented by the boldamino acids.

FIG. 20 shows the regions of similarity between the amino acid sequencesof the integrin alpha 11 subunit (a11) protein (SEQ ID NO:35) and thehuman integrin alpha 1 subunit (SEQ ID NO: 103).

FIG. 21 shows an analysis of the integrin alpha 11 subunit (a11) aminoacid sequence. Alpha, beta, turn and coil regions; hydrophilicity andhydrophobicity; amphipathic regions; flexible regions; antigenic indexand surface probability are shown.

Its translation product has homology to the characteristicimmunoglobulin and major histocompatibility complex protein domain ofintegrin family members. As shown in FIGS. 19A-F, a11 has transmembranedomains (the transmembrane domains comprise amino acids 666-682 and/or1145-1161 of SEQ ID NO:35; which correspond to amino acids 666-682and/or 1145-1161 of FIGS. 19A-F) with strong conservation between othermembers of the integrin family. The polynucleotide contains an openreading frame encoding the a11 polypeptide of 1189 amino acids. Thepresent invention exhibits a high degree of homology at the amino acidlevel to the human integrin alpha 1 subunit (as shown in FIG. 20).

Preferred polypeptides of the invention comprise, or alternativelyconsist of, the following amino acid sequence:TNGYQKTGDVYKCPVIHGNCTKLNLGRVT

LSNV (SEQ ID NO:102). Polynucleotides encoding these polypeptides arealso encompassed by the invention, as are antibodies that bind one ormore of these polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two,twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven,twenty-eight, twenty-nine, thirty, thirty-one, thirty-two, thirty-three,or all thirty-three of the immunogenic epitopes shown in SEQ ID NO: 35as residues: Phe-23 to Arg-31, Leu-62 to Asp-72, Val-96 to Asp-101,Thr-111 to Asn-116, Glu-128 to Thr-135, Val-142 to Ser-149, Asn-217 toVal-222, Glu-233 to Arg-241, Gly-272 to Leu-280, Gln-286 to Thr-293,Tyr-303 to Ile-308, Gly-354 to Thr-360, Glu-408 to Lys-419, Glu-508 toLys-514, Arg-521 to Val-526, Gly-529 to Phe-542, Asp-551 to Tyr-557,Thr-587 to Thr-593, His-656 to Asp-665, Met-697 to Arg-705, Asp-709 toThr-716, Glu-755 to Gly-760, Asn-779 to His-786, Leu-810 to Asp-816,Leu-844 to Ala-851, Gln-871 to Gly-877, Glu-884 to Gln-889, Ser-931 toAsn-943, Ser-974 to Ile-982, Gly-1039 to Gln-1058, Arg-1121 to Arg-1127,Ser-1134 to Trp-1139, and/or Ser-1172 to Pro-1183. Polynucleotidesencoding these polypeptides are also encompassed by the invention as areantibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g., fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the a11 polypeptide having theamino acid sequence shown in FIGS. 19A-F (SEQ ID NO:35). The nucleotidesequence shown in FIGS. 19A-F (SEQ ID NO:35) was obtained by sequencinga cloned cDNA (HOHBY69), which was deposited on Nov. 17 at the AmericanType Culture Collection, and given Accession Number 203484.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO:17 is intended DNA fragments atleast about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:17. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:17. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Representative examples of all polynucleotide fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, from about 501 toabout 550, from about 551 to about 600, from about 601 to about 650,from about 651 to about 700, from about 701 to about 750, from about 751to about 800, from about 801 to about 850, from about 851 to about 900,from about 901 to about 950, from about 951 to about 1000, from about1001 to about 1050, from about 1051 to about 1100, from about 1101 toabout 1150, from about 1151 to about 1200, from about 1201 to about1250, from about 1251 to about 1300, from about 1301 to about 1350, fromabout 1351 to about 1400, from about 1401 to about 1450, from about 1451to about 1500, from about 1501 to about 1550, from about 1551 to about1600, from about 1601 to about 1650, from about 1651 to about 1700, fromabout 1701 to about 1750, from about 1751 to about 1800, from about 1801to about 1850, from about 1851 to about 1900, from about 1901 to about1950, from about 1951 to about 2000, from about 2001 to about 2050, fromabout 2051 to about 2100, from about 2101 to about 2150, from about 2151to about 2200, from about 2201 to about 2250, from about 2251 to about2300, from about 2301 to about 2350, from about 2351 to about 2400, fromabout 2401 to about 2450, from about 2451 to about 2500, from about 2501to about 2550, from about 2551 to about 2600, from about 2601 to about2650, from about 2651 to about 2700, from about 2701 to about 2750, fromabout 2751 to about 2800, from about 2801 to about 2850, from about 2851to about 2900, from about 2901 to about 2950, from about 2951 to about3000, from about 3001 to about 3050, from about 3051 to about 3100, fromabout 3101 to about 3150, from about 3151 to about 3200, from about 3201to about 3250, from about 3251 to about 3300, from about 3301 to about3350, from about 3351 to about 3400, from about 3401 to about 3450, fromabout 3451 to about 3500, from about 3501 to about 3550, from about 3551to about 3600, from about 3601 to about 3650, from about 3651 to about3700, from about 3701 to about 3750, from about 3751 to about 3800, fromabout 3801 to about 3850, from about 3851 to about 3900, from about 3901to about 3950, from about 3951 to about 4000, from about 4001 to about4050, from about 4051 to about 4100, from about 4101 to about 4150, fromabout 4151 to about 4200, from about 4201 to about 4250, from about 4251to about 4300, from about 4301 to about 4350, from about 4351 to about4400, from about 4401 to about 4450, from about 4451 to about 4500, fromabout 4501 to about 4550, from about 4551 to about 4600, from about 4601to about 4650, from about 4651 to about 4700, from about 4701 to about4750, from about 4751 to about 4800, from about 4801 to about 4850, fromabout 4851 to about 4900, from about 4901 to about 4950, from about 4951to about 4995, from about, from about 1 to about 236, from about 144 toabout 188, from about 231 to about 276 of SEQ ID NO:17, or thecomplementary strand thereto, or the cDNA contained in the depositedgene. In this context “about” includes the particularly recited ranges,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding a member selected from the group: apolypeptide comprising or alternatively, consisting of, any one of thetransmembrane domains (amino acid residues from about 666 to about 682and/or 1145 to about 1161 in FIGS. 19A-F (amino acids from about 666 toabout 682 and/or 1145 to about 1161 in SEQ ID NO:35), in addition to theimmunoglobulin and major histocompatibility complex protein domain(amino acid residues from about 64 to about 96 in FIGS. 19A-F (aminoacids from about 64 to about 96 in SEQ ID NO:35). Since the location ofthese domains have been predicted by computer analysis, one of ordinaryskill would appreciate that the amino acid residues constituting thesedomains may vary slightly (e.g., by about 1 to 15 amino acid residues)depending on the criteria used to define each domain. In additionalembodiments, the polynucleotides of the invention encode functionalattributes of a11.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of the present invention.

The data representing the structural or functional attributes of a11 setforth in FIG. 21 and/or Table VII, as described above, was generatedusing the various modules and algorithms of the DNA*STAR set on defaultparameters. In a preferred embodiment, the data presented in columnsVIII, IX XIII, and XIV of Table VII can be used to determine regions ofa11 which exhibit a high degree of potential for antigenicity. Regionsof high antigenicity are determined from the data presented in columnsVIII, IX, XIII, and/or XIV by choosing values which represent regions ofthe polypeptide which are likely to be exposed on the surface of thepolypeptide in an environment in which antigen recognition may occur inthe process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 21, butmay, as shown in Table VII, be represented or identified by usingtabular representations of the data presented in FIG. 21. The DNA*STARcomputer algorithm used to generate FIG. 21 (set on the original defaultparameters) was used to present the data in FIG. 21 in a tabular format(See Table VII). The tabular format of the data in FIG. 21 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 21 and in Table VIIinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS.19A-F. As set out in FIG. 21 and in Table VII, such preferred regionsinclude Gamier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, etc.) may still beretained. For example, the ability of shortened a11 muteins to induceand/or bind to antibodies which recognize the complete or mature formsof the polypeptides generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the N-terminus. Whether a particular polypeptide lackingN-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that an a11 muteinwith a large number of deleted N-terminal amino acid residues may retainsome biological or immunogenic activities. In fact, peptides composed ofas few as six a11 amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the a11 aminoacid sequence shown in FIGS. 19A-F, up to the threonine residue atposition number 1184 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-1189 of FIGS. 19A-F, where n1 is aninteger from 2 to 1184 corresponding to the position of the amino acidresidue in FIGS. 19A-F (which is identical to the sequence shown as SEQID NO:35). In another embodiment, N-terminal deletions of the allpolypeptide can be described by the general formula n2-1189, where n2 isa number from 2 to 1184, corresponding to the position of amino acididentified in FIG. 19. N-terminal deletions of the a11 polypeptide ofthe invention shown as SEQ ID NO:35 include polypeptides comprising theamino acid sequence of residues: N-terminal deletions of the a11polypeptide of the invention shown as SEQ ID NO:35 include polypeptidescomprising the amino acid sequence of residues: D-2 to E-1189; L-3 toE-1189; P-4 to E-1189; R-5 to E-1189; G-6 to E-1189; L-7 to E-1189; V-9to E-1189; A-10 to E-1189; W-11 to E-1189; A-12 to E-1189; L-13 toE-1189; S-14 to E-1189; L-15 to E-1189; W-16 to E-1189; P-17 to E-1189;G-18 to E-1189; F-19 to E-1189; T-20 to E-1189; D-21 to E-1189; T-22 toE-1189; F-23 to E-1189; N-24 to E-1189; M-25 to E-1189; D-26 to E-1189;T-27 to E-1189; R-28 to E-1189; K-29 to E-1189; P-30 to E-1189; R-31 toE-1189; V-32 to E-1189; I-33 to E-1189; P-34 to E-1189; G-35 to E-1189;S-36 to E-1189; R-37 to E-1189; T-38 to E-1189; A-39 to E-1189; F-40 toE-1189; F-41 to E-1189; G-42 to E-1189; Y-43 to E-1189; T44 to E-1189;V-45 to E-1189; Q-46 to E-1189; Q-47 to E-1189; H-48 to E-1189; D-49 toE-1189; I-50 to E-1189; S-51 to E-1189; G-52 to E-1189; N-53 to E-1189;K-54 to E-1189; W-55 to E-1189; L-56 to E-1189; V-57 to E-1189; V-58 toE-1189; G-59 to E-1189; A-60 to E-1189; P-61 to E-1189; L-62 to E-1189;E-63 to E-1189; T-64 to E-1189; N-65 to E-1189; G-66 to E-1189; Y-67 toE-1189; Q-68 to E-1189; K-69 to E-1189; T-70 to E-1189; G-71 to E-1189;D-72 to E-1189; V-73 to E-1189; Y-74 to E-1189; K-75 to E-1189; C-76 toE-1189; P-77 to E-1189; V-78 to E-1189; I-79 to E-1189; H-80 to E-1189;G-81 to E-1189; N-82 to E-1189; C-83 to E-1189; T-84 to E-1189; K-85 toE-1189; L-86 to E-1189; N-87 to E-1189; L-88 to E-1189; G-89 to E-1189;R-90 to E-1189; V-91 to E-1189; T-92 to E-1189; L-93 to E-1189; S-94 toE-1189; N-95 to E-1189; V-96 to E-1189; S-97 to E-1189; E-98 to E-1189;R-99 to E-1189; K-100 to E-1189; D-101 to E-1189; N-102 to E-1189; M-103to E-1189; R-104 to E-1189; L-105 to E-1189; G-106 to E-1189; L-107 toE-1189; S-108 to E-1189; L-109 to E-1189; A-110 to E-1189; T-111 toE-1189; N-112 to E-1189; P-113 to E-1189; K-114 to E-1189; D-115 toE-1189; N-116 to E-1189; S-117 to E-1189; F-118 to E-1189; L-119 toE-1189; A-120 to E-1189; C-121 to E-1189; S-122 to E-1189; P-123 toE-1189; L-124 to E-1189; W-125 to E-1189; S-126 to E-1189; H-127 toE-1189; E-128 to E-1189; C-129 to E-1189; G-130 to E-1189; S-131toE-1189; S-132 to E-1189; Y-133 to E-1189; Y-134 to E-1189; T-135 toE-1189; T-136 to E-1189; G-137 to E-1189; M-138 to E-1189; C-139 toE-1189; S-140 to E-1189; R-141 to E-1189; V-142 to E-1189; N-143 toE-1189; S-144 to E-1189; N-145 to E-1189; F-146 to E-1189; R-147 toE-1189; F-148 to E-1189; S-149 to E-1189; K-150 to E-1189; T-151 toE-1189; V-152 to E-1189; A-153 to E-1189; P-154 to E-1189; A-155 toE-1189; L-156 to E-1189; Q-157 to E-1189; R-158 to E-1189; C-159 toE-1189; Q-160 to E-1189; T-161 to E-1189; Y-162 to E-1189; M-163 toE-1189; D-164 to E-1189; I-165 to E-1189; V-166 to E-1189; I-167 toE-1189; V-168 to E-1189; L-169 to E-1189; D-170 to E-1189; G-171 toE-1189; S-172 to E-1189; N-173 to E-1189; S-174 to E-1189; I-175 toE-1189; Y-176 to E-1189; P-177 to E-1189; W-178 to E-1189; V-179 toE-1189; E-180 to E-1189; V-181 to E-1189; Q-182 to E-1189; H-183 toE-1189; F-184 to E-1189; L-185 to E-1189; I-186 to E-1189; N-187 toE-1189; I-188 to E-1189; L-189 to E-1189; K-190 to E-1189; K-191 toE-1189; F-192 to E-1189; Y-193 to E-1189; I-194 to E-1189; G-195 toE-1189; P-196 to E-1189; G-197 to E-1189; Q-198 to E-1189; I-199 toE-1189; Q-200 to E-1189; V-201 to E-1189; G-202 to E-1189; V-203 toE-1189; V-204 to E-1189; Q-205 to E-1189; Y-206 to E-1189; G-207 toE-1189; E-208 to E-1189; D-209 to E-1189; V-210 to E-1189; V-211 toE-1189; H-212 to E-1189; E-213 to E-1189; F-214 to E-1189; H-215 toE-1189; L-216 to E-1189; N-217 to E-1189; D-218 to E-1189; Y-219 toE-1189; R-220 to E-1189; S-221 to E-1189; V-222 to E-1189; K-223 toE-1189; D-224 to E-1189; V-225 to E-1189; V-226 to E-1189; E-227 toE-1189; A-228 to E-1189; A-229 to E-1189; S-230 to E-1189; H-231 toE-1189; I-232 to E-1189; E-233 to E-1189; Q-234 to E-1189; R-235 toE-1189; G-236 to E-1189; G-237 to E-1189; T-238 to E-1189; E-239 toE-1189; T-240 to E-1189; R-241 to E-1189; T-242 to E-1189; A-243 toE-1189; F-244 to E-1189; G-245 to E-1189; I-246 to E-1189; E-247 toE-1189; F-248 to E-1189; A-249 to E-1189; R-250 to E-1189; S-251 toE-1189; E-252 to E-1189; A-253 to E-1189; F-254 to E-1189; Q-255 toE-1189; K-256 to E-1189; G-257 to E-1189; G-258 to E-1189; R-259 toE-1189; K-260 to E-1189; G-261 to E-1189; A-262 to E-1189; K-263 toE-1189; K-264 to E-1189; V-265 to E-1189; M-266 to E-1189; I-267 toE-1189; V-268 to E-1189; I-269 to E-1189; T-270 to E-1189; D-271 toE-1189; G-272 to E-1189; E-273 to E-1189; S-274 to E-1189; H-275 toE-1189; D-276 to E-1189; S-277 to E-1189; P-278 to E-1189; D-279 toE-1189; L-280 to E-1189; E-281 to E-1189; K-282 to E-1189; V-283 toE-1189; I-284 to E-1189; Q-285 to E-1189; Q-286 to E-1189; S-287 toE-1189; E-288 to E-1189; R-289 to E-1189; D-290 to E-1189; N-291 toE-1189; V-292 to E-1189; T-293 to E-1189; R-294 to E-1189; Y-295 toE-1189; A-296 to E-1189; V-297 to E-1189; A-298 to E-1189; V-299 toE-1189; L-300 to E-1189; G-301 to E-1189; Y-302 to E-1189; Y-303 toE-1189; N-304 to E-1189; R-305 to E-1189; R-306 to E-1189; G-307 toE-1189; I-308 to E-1189; N-309 to E-1189; P-310 to E-1189; E-311 toE-1189; T-312 to E-1189; F-313 to E-1189; L-314 to E-1189; N-315 toE-1189; E-316 to E-1189; I-317 to E-1189; K-318 to E-1189; Y-319 toE-1189; I-320 to E-1189; A-321 to E-1189; S-322 to E-1189; D-323 toE-1189; P-324 to E-1189; D-325 to E-1189; D-326 to E-1189; K-327 toE-1189; H-328 to E-1189; F-329 to E-1189; F-330 to E-1189; N-331 toE-1189; V-332 to E-1189; T-333 to E-1189; D-334 to E-1189; E-335 toE-1189; A-336 to E-1189; A-337 to E-1189; L-338 to E-1189; K-339 toE-1189; D-340 to E-1189; I-341 to E-1189; V-342 to E-1189; D-343 toE-1189; A-344 to E-1189; L-345 to E-1189; G-346 to E-1189; D-347 toE-1189; R-348 to E-1189; I-349 to E-1189; F-350 to E-1189; S-351 toE-1189; L-352 to E-1189; E-353 to E-1189; G-354 to E-1189; T-355 toE-1189; N-356 to E-1189; K-357 to E-1189; N-358 to E-1189; E-359 toE-1189; T-360 to E-1189; S-361 to E-1189; F-362 to E-1189; G-363 toE-1189; L-364 to E-1189; E-365 to E-1189; M-366 to E-1189; S-367 toE-1189; Q-368 to E-1189; T-369 to E-1189; G-370 to E-1189; F-371 toE-1189; S-372 to E-1189; S-373 to E-1189; H-374 to E-1189; V-375 toE-1189; V-376 to E-1189; E-377 to E-1189; D-378 to E-1189; G-379 toE-1189; V-380 to E-1189; L-381 to E-1189; L-382 to E-1189; G-383 toE-1189; A-384 to E-1189; V-385 to E-1189; G-386 to E-1189; A-387 toE-1189; Y-388 to E-1189; D-389 to E-1189; W-390 to E-1189; N-391 toE-1189; G-392 to E-1189; A-393 to E-1189; V-394 to E-1189; L-395 toE-1189; K-396 to E-1189; E-397 to E-1189; T-398 to E-1189; S-399 toE-1189; A-400 to E-1189; G-401 to E-1189; K-402 to E-1189; V-403 toE-1189; I-404 to E-1189; P-405 to E-1189; L-406 to E-1189; R-407 toE-1189; E-408 to E-1189; S-409 to E-1189; Y-410 to E-1189; L-411 toE-1189; K-412 to E-1189; E-413 to E-1189; F-414 to E-1189; P-415 toE-1189; E-416 to E-1189; E-417 to E-1189; L-418 to E-1189; K-419 toE-1189; N-420 to E-1189; H-421 to E-1189; G-422 to E-1189; A-423 toE-1189; Y-424 to E-1189; L-425 to E-1189; G-426 to E-1189; Y-427 toE-1189; T-428 to E-1189; V-429 to E-1189; T-430 to E-1189; S-431 toE-1189; V-432 to E-1189; V-433 to E-1189; S-434 to E-1189; S-435 toE-1189; R-436 to E-1189; Q-437 to E-1189; G-438 to E-1189; R-439 toE-1189; V-440 to E-1189; Y-441 to E-1189; V-442 to E-1189; A-443 toE-1189; G-444 to E-1189; A-445 to E-1189; P-446 to E-1189; R-447 toE-1189; F-448 to E-1189; N-449 to E-1189; H-450 to E-1189; T-451 toE-1189; G-452 to E-1189; K-453 to E-1189; V-454 to E-1189; I-455 toE-1189; L-456 to E-1189; F-457 to E-1189; T-458 to E-1189; M-459 toE-1189; H-460 to E-1189; N-461 to E-1189; N-462 to E-1189; R-463 toE-1189; S-464 to E-1189; L-465 to E-1189; T-466 to E-1189; I-467 toE-1189; H-468 to E-1189; Q-469 to E-1189; A-470 to E-1189; M-471 toE-1189; R-472 to E-1189; G-473 to E-1189; Q-474 to E-1189; Q-475 toE-1189; I-476 to E-1189; G-477 to E-1189; S-478 to E-1189; Y-479 toE-1189; F-480 to E-1189; G-481 to E-1189; S-482 to E-1189; E-483 toE-1189; I-484 to E-1189; T-485 to E-1189; S-486 to E-1189; V-487 toE-1189; D-488 to E-1189; I-489 to E-1189; D-490 to E-1189; G-491 toE-1189; D-492 to E-1189; G-493 to E-1189; V-494 to E-1189; T-495 toE-1189; D-496 to E-1189; V-497 to E-1189; L-498 to E-1189; L-499 toE-1189; V-500 to E-1189; G-501 to E-1189; A-502 to E-1189; P-503 toE-1189; M-504 to E-1189; Y-505 to E-1189; F-506 to E-1189; N-507 toE-1189; E-508 to E-1189; G-509 to E-1189; R-510 to E-1189; E-511 toE-1189; R-512 to E-1189; G-513 to E-1189; K-514 to E-1189; V-515 toE-1189; Y-516 to E-1189; V-517 to E-1189; Y-518 to E-1189; E-519 toE-1189; L-520 to E-1189; R-521 to E-1189; Q-522 to E-1189; N-523 toE-1189; R-524 to E-1189; F-525 to E-1189; V-526 to E-1189; Y-527 toE-1189; N-528 to E-1189; G-529 to E-1189; T-530 to E-1189; L-531 toE-1189; K-532 to E-1189; D-533 to E-1189; S-534 to E-1189; H-535 toE-1189; S-536 to E-1189; Y-537 to E-1189; Q-538 to E-1189; N-539 toE-1189; A-540 to E-1189; R-541 to E-1189; F-542 to E-1189; G-543 toE-1189; S-544 to E-1189; S-545 to E-1189; I-546 to E-1189; A-547 toE-1189; S-548 to E-1189; V-549 to E-1189; R-550 to E-1189; D-551 toE-1189; L-552 to E-1189; N-553 to E-1189; Q-554 to E-1189; D-555 toE-1189; S-556 to E-1189; Y-557 to E-1189; N-558 to E-1189; D-559 toE-1189; V-560 to E-1189; V-561 to E-1189; V-562 to E-1189; G-563 toE-1189; A-564 to E-1189; P-565 to E-1189; L-566 to E-1189; E-567 toE-1189; D-568 to E-1189; N-569 to E-1189; H-570 to E-1189; A-571 toE-1189; G-572 to E-1189; A-573 to E-1189; I-574 to E-1189; Y-575 toE-1189; I-576 to E-1189; F-577 to E-1189; H-578 to E-1189; G-579 toE-1189; F-580 to E-1189; R-581 to E-1189; G-582 to E-1189; S-583 toE-1189; I-584 to E-1189; L-585 to E-1189; K-586 to E-1189; T-587 toE-1189; P-588 to E-1189; K-589 to E-1189; Q-590 to E-1189; R-591 toE-1189; I-592 to E-1189; T-593 to E-1189; A-594 to E-1189; S-595 toE-1189; E-596 to E-1189; L-597 to E-1189; A-598 to E-1189; T-599 toE-1189; G-600 to E-1189; L-601 to E-1189; Q-602 to E-1189; Y-603 toE-1189; F-604 to E-1189; G-605 to E-1189; C-606 to E-1189; S-607 toE-1189; I-608 to E-1189; H-609 to E-1189; G-610 to E-1189; Q-611 toE-1189; L-612 to E-1189; D-613 to E-1189; L-614 to E-1189; N-615 toE-1189; E-616 to E-1189; D-617 to E-1189; G-618 to E-1189; L-619 toE-1189; I-620 to E-1189; D-621 to E-1189; L-622 to E-1189; A-623 toE-1189; V-624 to E-1189; G-625 to E-1189; A-626 to E-1189; L-627 toE-1189; G-628 to E-1189; N-629 to E-1189; A-630 to E-1189; V-631 toE-1189; I-632 to E-1189; L-633 to E-1189; W-634 to E-1189; S-635 toE-1189; R-636 to E-1189; P-637 to E-1189; V-638 to E-1189; V-639 toE-1189; Q-640 to E-1189; I-641 to E-1189; N-642 to E-1189; A-643 toE-1189; S-644 to E-1189; L-645 to E-1189; H-646 to E-1189; F-647 toE-1189; E-648 to E-1189; P-649 to E-1189; S-650 to E-1189; K-651 toE-1189; I-652 to E-1189; N-653 to E-1189; I-654 to E-1189; F-655 toE-1189; H-656 to E-1189; R-657 to E-1189; D-658 to E-1189; C-659 toE-1189; K-660 to E-1189; R-661 to E-1189; S-662 to E-1189; G-663 toE-1189; R-664 to E-1189; D-665 to E-1189; A-666 to E-1189; T-667 toE-1189; C-668 to E-1189; L-669 to E-1189; A-670 to E-1189; A-671 toE-1189; F-672 to E-1189; L-673 to E-1189; C-674 to E-1189; F-675 toE-1189; T-676 to E-1189; P-677 to E-1189; I-678 to E-1189; F-679 toE-1189; L-680 to E-1189; A-681 to E-1189; P-682 to E-1189; H-683 toE-1189; F-684 to E-1189; Q-685 to E-1189; T-686 to E-1189; T-687 toE-1189; T-688 to E-1189; V-689 to E-1189; G-690 to E-1189; I-691 toE-1189; R-692 to E-1189; Y-693 to E-1189; N-694 to E-1189; A-695 toE-1189; T-696 to E-1189; M-697 to E-1189; D-698 to E-1189; E-699 toE-1189; R-700 to E-1189; R-701 to E-1189; Y-702 to E-1189; T-703 toE-1189; P-704 to E-1189; R-705 to E-1189; A-706 to E-1189; H-707 toE-1189; L-708 to E-1189; D-709 to E-1189; E-710 to E-1189; G-711 toE-1189; G-712 to E-1189; D-713 to E-1189; R-714 to E-1189; F-715 toE-1189; T-716 to E-1189; N-717 to E-1189; R-718 to E-1189; A-719 toE-1189; V-720 to E-1189; L-721 to E-1189; L-722 to E-1189; S-723 toE-1189; S-724 to E-1189; G-725 to E-1189; Q-726 to E-1189; E-727 toE-1189; L-728 to E-1189; C-729 to E-1189; E-730 to E-1189; R-731 toE-1189; I-732 to E-1189; N-733 to E-1189; F-734 to E-1189; H-735 toE-1189; V-736 to E-1189; L-737 to E-1189; D-738 to E-1189; T-739 toE-1189; A-740 to E-1189; D-741 to E-1189; Y-742 to E-1189; V-743 toE-1189; K-744 to E-1189; P-745 to E-1189; V-746 to E-1189; T-747 toE-1189; F-748 to E-1189; S-749 to E-1189; V-750 to E-1189; E-751 toE-1189; Y-752 to E-1189; S-753 to E-1189; L-754 to E-1189; E-755 toE-1189; D-756 to E-1189; P-757 to E-1189; D-758 to E-1189; H-759 toE-1189; G-760 to E-1189; P-761 to E-1189; M-762 to E-1189; L-763 toE-1189; D-764 to E-1189; D-765 to E-1189; G-766 to E-1189; W-767 toE-1189; P-768 to E-1189; T-769 to E-1189; T-770 to E-1189; L-771 toE-1189; R-772 to E-1189; V-773 to E-1189; S-774 to E-1189; V-775 toE-1189; P-776 to E-1189; F-777 to E-1189; W-778 to E-1189; N-779 toE-1189; G-780 to E-1189; C-781 to E-1189; N-782 to E-1189; E-783 toE-1189; D-784 to E-1189; E-785 to E-1189; H-786 to E-1189; C-787 toE-1189; V-788 to E-1189; P-789 to E-1189; D-790 to E-1189; L-791 toE-1189; V-792 to E-1189; L-793 to E-1189; D-794 to E-1189; A-795 toE-1189; R-796 to E-1189; S-797 to E-1189; D-798 to E-1189; L-799 toE-1189; P-800 to E-1189; T-801 to E-1189; A-802 to E-1189; M-803 toE-1189; E-804 to E-1189; Y-805 to E-1189; C-806 to E-1189; Q-807 toE-1189; R-808 to E-1189; V-809 to E-1189; L-810 to E-1189; R-811 toE-1189; K-812 to E-1189; P-813 to E-1189; A-814 to E-1189; Q-815 toE-1189; D-816 to E-1189; C-817 to E-1189; S-818 to E-1189; A-819 toE-1189; Y-820 to E-1189; T-821 to E-1189; L-822 to E-1189; S-823 toE-1189; F-824 to E-1189; D-825 to E-1189; T-826 to E-1189; T-827 toE-1189; V-828 to E-1189; F-829 to E-1189; I-830 to E-1189; I-831 toE-1189; E-832 to E-1189; S-833 to E-1189; T-834 to E-1189; R-835 toE-1189; Q-836 to E-1189; R-837 to E-1189; V-838 to E-1189; A-839 toE-1189; V-840to E-1189; E-841 to E-1189; A-842 to E-1189; T-843 toE-1189; L-844 to E-1189; E-845 to 1189; N-846 to E-1189; R-847 toE-1189; G-848 to E-1189; E-849 to E-1189; N-850 to E-1189; A-851 toE-1189; Y-852 to E-1189; S-853 to E-1189; T-854 to E-1189; V-855 toE-1189; L-856 to E-1189; N-857 to E-1189; I-858 to E-1189; S-859 toE-1189; Q-860 to E-1189; S-861 to E-1189; A-862 to E-1189; N-863 toE-1189; L-864 to E-1189; Q-865 to E-1189; F-866 to E-1189; A-867 toE-1189; S-868 to E-1189; L-869 to E-1189; I-870 to E-1189; Q-871 toE-1189; K-872 to E-1189; E-873 to E-1189; D-874 to E-1189; S-875 toE-1189; D-876 to E-1189; G-877 to E-1189; S-878 to E-1189; I-879 toE-1189; E-880 to E-1189; C-881 to E-1189; V-882 to E-1189; N-883 toE-1189; E-884 to E-1189; E-885 to E-1189; R-886 to E-1189; R-887 toE-1189; L-888 to E-1189; Q-889 to E-1189; K-890 to E-1189; Q-891 toE-1189; V-892 to E-1189; C-893 to E-1189; N-894 to E-1189; V-895 toE-1189; S-896 to E-1189; Y-897 to E-1189; P-898 to E-1189; F-899 toE-1189; F-900 to E-1189; R-901 to E-1189; A-902 to E-1189; K-903 toE-1189; A-904 to E-1189; K-905 to E-1189; V-906 to E-1189; A-907 toE-1189; F-908 to E-1189; R-909 to E-1189; L-910 to E-1189; D-911 toE-1189; F-912 to E-1189; E-913 to E-1189; F-914 to E-1189; S-915 toE-1189; K-916 to E-1189; S-917 to E-1189; I-918 to E-1189; F-919 toE-1189; L-920 to E-1189; H-921 to E-1189; H-922 to E-1189; L-923 toE-1189; E-924 to E-1189; I-925 to E-1189; E-926 to E-1189; L-927 toE-1189; A-928 to E-1189; A-929 to E-1189; G-930 to E-1189; S-931 toE-1189; D-932 to E-1189; S-933 to E-1189; N-934 to E-1189; E-935 toE-1189; R-936 to E-1189; D-937 to E-1189; S-938 to E-1189; T-939 toE-1189; K-940 to E-1189; E-941 to E-1189; D-942 to E-1189; N-943 toE-1189; V-944 to E-1189; A-945 to E-1189; P-946 to E-1189; L-947 toE-1189; R-948 to E-1189; F-949 to E-1189; H-950 to E-1189; L-951 toE-1189; K-952 to E-1189; Y-953 to E-1189; E-954 to E-1189; A-955 toE-1189; D-956 to E-1189; V-957 to E-1189; L-958 to E-1189; F-959 toE-1189; T-960 to E-1189; R-961 to E-1189; S-962 to E-1189; S-963 toE-1189; S-964 to E-1189; L-965 to E-1189; S-966 to E-1189; H-967 toE-1189; Y-968 to E-1189; E-969 to E-1189; V-970 to E-1189; K-971 toE-1189; L-972 to E-1189; N-973 to E-1189; S-974 to E-1189; S-975 toE-1189; L-976 to E-1189; E-977 to E-1189; R-978 to E-1189; Y-979 toE-1189; D-980 to E-1189; G-981 to E-1189; I-982 to E-1189; G-983 toE-1189; P-984 to E-1189; P-985 to E-1189; F-986 to E-1189; S-987 toE-1189; C-988 to E-1189; I-989 to E-1189; F-990 to E-1189; R-991 toE-1189; I-992 to E-1189; Q-993 to E-1189; N-994 to E-1189; L-995 toE-1189; G-996 to E-1189; L-997 to E-1189; F-998 to E-1189; P-999 toE-1189; I-1000 to E-1189; H-1001 to E-1189; G-1002 to E-1189; I-1003 toE-1189; M-1004 to E-1189; M-1005 to E-1189; K-1006 E-1189; I-1007 toE-1189; T-1008 to E-1189; I-1009 to E-1189; P-1010 to E-1189; I-1011 toE-1189; A-1012 to E-1189; T-1013 to E-1189; R-1014 to E-1189; S-1015 toE-1189; G-1016 to E-1189; N-1017 to E-1189; R-1018 to E-1189; L-1019 toE-1189; L-1020 to E-1189; K-1021 to E-1189; L-1022 to E-1189; R-1023 toE-1189; D-1024 to E-1189; F-1025 to E-1189; L-1026 to E-1189; T-1027 toE-1189; D-1028 to E-1189; E-1029 to E-1189; V-1030 to E-1189; A-1031 toE-1189; N-1032 to E-1189; T-1033 to E-1189; S-1034 to E-1189; C-1035 toE-1189; N-1036 to E-1189; I-1037 to E-1189; W-1038 to E-1189; G-1039 toE-1189; N-1040 to E-1189; S-1041 to E-1189; T-1042 to E-1189; E-1043 toE-1189; Y-1044 to E-1189; R-1045 to E-1189; P-1046 to E-1189; T-1047 toE-1189; P-1048 to E-1189; V-1049 to E-1189; E-1050 to E-1189; E-1051 toE-1189; D-1052 to E-1189; L-1053 to E-1189; R-1054 to E-1189; R-1055 toE-1189; A-1056 to E-1189; P-1057 to E-1189; Q-1058 to E-1189; L-1059 toE-1189; N-1060 to E-1189; H-1061 to E-1189; S-1062 to E-1189; N-1063toE-1189; S-1064to E-1189; D-1065 to E-1189; V-1066 to E-1189; V-1067 toE-1189; S-1068 to E-1189; I-1069 to E-1189; N-1070 to E-1189; C-1071 toE-1189; N-1072 to E-1189; I-1073 to E-1189; R-1074 to E-1189; L-1075 toE-1189; V-1076 to E-1189; P-1077 to E-1189; N-1078 to E-1189; Q-1079 toE-1189; E-1080 to E-1189; I-1081 to E-1189; N-1082 to E-1189; F-1083 toE-1189; H-1084 to E-1189; L-1085 to E-1189; L-1086 to E-1189; G-1087 toE-1189; N-1088 to E-1189; L-1089 to E-1189; W-1090to E-1189; L-1091 toE-1189; R-1092 to E-1189; S-1093 to E-1189; L-1094 to E-1189; K-1095 toE-1189; A-1096 to E-1189; L-1097 to E-1189; K-1098 to E-1189; Y-1099 toE-1189; K-1100 to E-1189; S-1101 to E-1189; M-1102 to E-1189; K-1103 toE-1189; I-1104 to E-1189; M-1105 to E-1189; V-1106 to E-1189; N-1107 toE-1189; A-1108 to E-1189; A-1109 to E-1189; L-1110to E-1189; Q-1111 toE-1189; R-1112 to E-1189; Q-1113 to E-1189; F-1114 to E-1189; H-1115 toE-1189; S-1116 to E-1189; P-1117 to E-1189; F-1118 to E-1189; I-1119 toE-1189; F-1120 to E-1189; R-1121 to E-1189; E-1122 to E-1189; E-1123 toE-1189; D-1124 to E-1189; P-1125 to E-1189; S-1126 to E-1189; R-1127 toE-1189; Q-1128 to E-1189; I-1129 to E-1189; V-1130 to E-1189; F-1131 toE-1189; E-1132 to E-1189; I-1133 to E-1189; S-1134 to E-1189; K-1135 toE-1189; Q-1136 to E-1189; E-1137 to E-1189; D-1138 to E-1189; W-1139 toE-1189; Q-1140 to E-1189; V-1141 to E-1189; P-1142 to E-1189; I-1143 toE-1189; W-1144 to E-1189; I-1145 to E-1189; I-1146 to E-1189; V-1147 toE-1189; G-1148 to E-1189; S-1149 to E-1189; T-1150 to E-1189; L-1151 toE-1189; G-1152 to E-1189; G-1153 to E-1189; L-1154 to E-1189; L-1155 toE-1189; L-1156 to E-1189; L-1157 to E-1189; A-1158 to E-1189; L-1159 toE-1189; L-1160 to E-1189; V-1161 to E-1189; L-1162 to E-1189; A-1163 toE-1189; L-1164 to E-1189; W-1165 to E-1189; K-1166 to E-1189; L-1167 toE-1189; G-1168 to E-1189; F-1169 to E-1189; F-1170 to E-1189; R-1171 toE-1189; S-1172 to E-1189; A-1173 to E-1189; R-1174 to E-1189; R-1175 toE-1189; R-1176 to E-1189; R-1177 to E-1189; E-1178 to E-1189; P-1179 toE-1189; G-1180 to E-1189; L-1181 to E-1189; D-1182 to E-1189; P-1183 toE-1189; T-1184 to E-1189; of SEQ ID NO:35. Polynucleotides encodingthese polypeptides are also encompassed by the invention, as areantibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g., fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities (e.g., ability to illicit mitogenicactivity, induce differentiation of normal or malignant cells, abilityto multimerize, etc.) may still be retained. For example the ability ofthe shortened a11 mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an a11 mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as six a11amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the all polypeptide shown in FIGS. 19A-F, up to the glycineresidue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m1 of FIGS. 19A-F,where m1 is an integer from 6 to 1189 corresponding to the position ofthe amino acid residue in FIGS. 19A-F. Moreover, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of C-terminal deletions of the alI polypeptide of the invention shown as SEQ ID NO:35 includepolypeptides comprising the amino acid sequence of residues: M-1 toL-1188; M-1 to V-1187; M-1 to K-1186; M-1 to P-1185; M-1 to T-1184; M-1to P-1183; M-1 to D-1182; M-1 to L-1181; M-1 to G-1180; M-1 to P-1179;M-1 to E-1178; M-1 to R-1177; M-1 to R-1176; M-1 to R-1175; M-1 toR-1174; M-1 to A-1173; M-1 to S-1172; M-1 to R-1171; M-1 to F-1170; M-1to F-1169; M-1 to G-1168; M-1 to L-1167; M-1 to K-1166; M-1 to W-1165;M-1 to L-1164; M-1 to A-1163; M-1 to L-1162; M-1 to V-1161; M-1 toL-1160; M-1 to L-1159; M-1 to A-1158; M-1 to L-1157; M-1 to L-1156; M-1to L-1155; M-1 to L-1154; M-1 to G-1153; M-1 to G-1152; M-1 to L-1151;M-1 to T-1150; M-1 to S-1149; M-1 to G-1148; M-1 to V-1147; M-1 toI-1146; M-1 to I-1145; M-1 to W-1144; M-1 to I-1143; M-1 to P-1142; M-1to V-1141; M-1 to Q-1140; M-1 to W-1139; M-1 to D-1138; M-1 to E-1137;M-1 to Q-1136; M-1 to K-1135; M-1 to S-1134; M-1 to I-1133; M-1 toE-1132; M-1 to F-1131; M-1 to V-1130; M-1 to I-1129; M-1 to Q-1128; M-1to R-1127; M-1 to S-1126; M-1 to P-1125; M-1 to D-1124; M-1 to E-1123;M-1 to E-1122; M-1 to R-1121; M-1 to F-1120; M-1 to I-1119; M-1 toF-1118; M-1 to P-1117; M-1 to S-1116; M-1 to H-1115; M-1 to F-1114; M-1to Q-1113; M-1 to R-1112; M-1 to Q-1111; M-1 to L-1110; M-1 to A-1109;M-1 to A-1108; M-1 to N-1107; M-1 to V-1106; M-1 to M-1105; M-1 toI-1104; M-1 to K-1103; M-1 to M-1102; M-1 to S-1101; M-1 to K-1100; M-1to Y-1099; M-1 to K-1098; M-1 to L-1097; M-1 to A-1096; M-1 to K-1095;M-1 to L-1094; M-1 to S-1093; M-1 to R-1092; M-1 to L-1091; M-1 toW-1090; M-1 to L-1089; M-1 to N-1088; M-1 to G-1087; M-1 to L-1086; M-1to L-1085; M-1 to H-1084; M-1 to F-1083; M-1 to N-1082; M-1 to I-1081;M-1 to E-1080; M-1 to Q-1079; M-1 to N-1078; M-1 to P-1077; M-1 toV-1076; M-1 to L-1075; M-1 to R-1074; M-1 to I-1073; M-1 to N-1072; M-1to C-1071; M-1 to N-1070; M-1 to I-1069; M-1 to S-1068; M-1 to V-1067;M-1 to V-1066; M-1 to D-1065; M-1 to S-1064; M-1 to N-1063; M-1 toS-1062; M-1 to H-1061; M-1 to N-1060; M-1 to L-1059; M-1 to Q-1058; M-1to P-1057; M-1 to A-1056; M-1 to R-1055; M-1 to R-1054; M-1 to L-1053;M-1 to D-1052; M-1 to E-1051; M-1 to E-1050; M-1 to V-1049; M-1 toP-1048; M-1 to T-1047; M-1 to P-1046; M-1 to R-1045; M-1 to Y-1044; M-1to E-1043; M-1 to T-1042; M-1 to S-1041; M-1 to N-1040; M-1 to G-1039;M-1 to W-1038; M-1 to I-1037; M-1 to N-1036; M-1 to C-1035; M-1 toS-1034; M-1 to T-1033; M-1 to N-1032; M-1 to A-1031; M-1 to V-1030; M-1to E-1029; M-1 to D-1028; M-1 to T-1027; M-1 to L-1026; M-1 to F-1025;M-1 to D-1024; M-1 to R-1023; M-1 to L-1022; M-1 to K-1021; M-1 toL-1020; M-1 to L-1019; M-1 to R-1018; M-1 to N-1017; M-1 to G-1016; M-1to S-1015; M-1 to R-1014; M-1 to T-1013; M-1 to A-1012; M-1 to I-1011;M-1 to P-1010; M-1 to I-1009; M-1 to T-1008; M-1 to I-1007; M-1 toK-1006; M-1 to M-1005; M-1 to M-1004; M-1 to I-1003; M-1 to G-1002; M-1to H-1001; M-1 to I-1000; M-1 to P-999; M-1 to F-998; M-1 to L-997; M-1to G-996; M-1 to L-995; M-1 to N-994; M-1 to Q-993; M-1 to I-992; M-1 toR-991; M-1 to F-990; M-1 to I-989; M-1 to C-988; M-1 to S-987; M-1 toF-986; M-1 to P-985; M-1 to P-984; M-1 to G-983; M-1 to I-982; M-1 toG-981; M-1 to D-980; M-1 to Y-979; M-1 to R-978; M-1 to E-977; M-1 toL-976; M-1 to S-975; M-1 to S-974; M-1 to N-973; M-1 to L-972; M-1 toK-971; M-1 to V-970; M-1 to E-969; M-1 to Y-968; M-1 to H-967; M-1 toS-966; M-1 to L-965; M-1 to S-964; M-1 to S-963; M-1 to S-962; M-1 toR-961; M-1 to T-960; M-1 to F-959; M-1 to L-958; M-1 to V-957; M-1 toD-956; M-1 to A-955; M-1 to E-954; M-1 to Y-953; M-1 to K-952; M-1 toL-951; M-1 to H-950; M-1 to F-949; M-1 to R-948; M-1 to L-947; M-1 toA-946; M-1 to A-945; M-1 to V-944; M-1 to N-943; M-1 to D-942; M-1 toE-941; M-1 to K-940; M-1 to T-939; M-1 to S-938; M-1 to D-937; M-1 toR-936; M-1 to E-935; M-1 to N-934; M-1 to S-933; M-1 to D-932; M-1 toS-931; M-1 to G-930; M-1 to A-929; M-1 to A-928; M-1 to L-927; M-1 toE-926; M-1 to I-925; M-1 to E-924; M-1 to L-923; M-1 to H-922; M-1 toH-921; M-1 to L-920; M-1 to F-919; M-1 to I-918; M-1 to S-917; M-1 toK-916; M-1 to S-915; M-1 to F-914; M-1 to E-913; M-1 to F-912; M-1 toD-911; M-1 to L-910; M-1 to R-909; M-1 to F-908; M-1 to A-907; M-1 toV-906; M-1 to K-905; M-1 to A-904; M-1 to K-903; M-1 to A-902; M-1 toR-901; M-1 to F-900; M-1 to F-899; M-1 to P-898; M-1 to Y-897; M-1 toS-896; M-1 to V-895; M-1 to N-894; M-1 to C-893; M-1 to V-892; M-1 toQ-891; M-1 to K-890; M-1 to Q-889; M-1 to L-888; M-1 to R-887; M-1 toR-886; M-1 to E-885; M-1 to E-884; M-1 to N-883; M-1 to V-882; M-1 toC-881; M-1 to E-880; M-1 to I-879; M-1 to S-878; M-1 to G-877; M-1 toD-876; M-1 to S-875; M-1 to D-874; M-1 to E-873; M-1 to K-872; M-1 toQ-871; M-1 to I-870; M-1 to L-869; M-1 to S-868; M-1 to A-867; M-1 toF-866; M-1 to Q-865; M-1 to L-864; M-1 to N-863; M-1 to A-862; M-1 toS-861; M-1 to Q-860; M-1 to S-859; M-1 to I-858; M-1 to N-857; M-1 toL-856; M-1 to V-855; M-1 to T-854; M-1 to S-853; M-1 to Y-852; M-1 toA-851; M-1 to N-850; M-1 to E-849; M-1 to G-848; M-1 to R-847; M-1 toN-846; M-1 to E-845; M-1 to L-844; M-1 to T-843; M-1 to A-842; M-1 toE-841; M-1 to V-840; M-1 to A-839; M-1 to V-838; M-1 to R-837; M-1 toQ-836; M-1 to R-835; M-1 to T-834; M-1 to S-833; M-1 to E-832; M-1 toI-831; M-1 to I-830; M-1 to F-829; M-1 to V-828; M-1 to T-827; M-1 toT-826; M-1 to D-825; M-1 to F-824; M-1 to S-823; M-1 to L-822; M-1 toT-821; M-1 to Y-820; M-1 to A-819; M-1 to S-818; M-1 to C-817; M-1 toD-816; M-1 to Q-815; M-1 to A-814; M-1 to P-813; M-1 to K-812; M-1 toR-811; M-1 to L-810; M-1 to V-809; M-1 to R-808; M-1 to Q-807; M-1 toC-806; M-1 to Y-805; M-1 to E-804; M-1 to M-803; M-1 to A-802; M-1 toT-801; M-1 to P-800; M-1 to L-799; M-1 to D-798; M-1 to S-797; M-1 toR-796; M-1 to A-795; M-1 to D-794; M-1 to L-793; M-1 to V-792; M-1 toL-791; M-1 to D-790; M-1 to P-789; M-1 to V-788; M-1 to C-787; M-1 toH-786; M-1 to E-785; M-1 to D-784; M-1 to E-783; M-1 to N-782; M-1 toC-781; M-1 to G-780; M-1 to N-779; M-1 to W-778; M-1 to F-777; M-1 toP-776; M-1 to V-775; M-1 to S-774; M-1 to V-773; M-1 to R-772; M-1 toL-771; M-1 to T-770; M-1 to T-769; M-1 to P-768; M-1 to W-767; M-1 toG-766; M-1 to D-765; M-1 to D-764; M-1 to L-763; M-1 to M-762; M-1 toP-761; M-1 to G-760; M-1 to H-759; M-1 to D-758; M-1 to P-757; M-1 toD-756; M-1 to E-755; M-1 to L-754; M-1 to S-753; M-1 to Y-752; M-1 toE-751; M-1 to V-750; M-1 to S-749; M-1 to F-748; M-1 to T-747; M-1 toV-746; M-1 to P-745; M-1 to K-744; M-1 to V-743; M-1 to Y-742; M-1 toD-741; M-1 to A-740; M-1 to T-739; M-1 to D-738; M-1 to L-737; M-1 toV-736; M-1 to H-735; M-1 to F-734; M-1 to N-733; M-1 to I-732; M-1 toR-731; M-1 to E-730; M-1 to C-729; M-1 to L-728; M-1 to E-727; M-1 toQ-726; M-1 to G-725; M-1 to S-724; M-1 to S-723; M-1 to L-722; M-1 toL-721; M-1 to V-720; M-1 to A-719; M-1 to R-718; M-1 to N-717; M-1 toT-716; M-1 to F-715; M-1 to R-714; M-1 to D-713; M-1 to G-712; M-1 toG-711; M-1 to E-710; M-1 to D-709; M-1 to L-708; M-1 to H-707; M-1 toA-706; M-1 to R-705; M-1 to P-704; M-1 to T-703; M-1 to Y-702; M-1 toR-701; M-1 to R-700; M-1 to E-699; M-1 to D-698; M-1 to M-697; M-1 toT-696; M-1 to A-695; M-1 to N-694; M-1 to Y-693; M-1 to R-692; M-1 toI-691; M-1 to G-690; M-1 to V-689; M-1 T-688; M-1 to T-687; M-1 toT-686; M-1 to Q-685; M-1 to F-684; M-1 to H-683; M-1 to P-682; M-1 toA-681; M-1 to L-680; M-1 to F-679; M-1 to I-678; M-1 to P-677; M-1 toT-676; M-1 to F-675; M-1 to C-674; M-1 to L-673; M-1 to F-672; M-1 toA-671; M-1 to A-670; M-1 to L-669; M-1 to C-668; M-1 to T-667; M-1 toA-666; M-1 to D-665; M-1 to R-664; M-1 to G-663; M-1 to S-662; M-1 toR-661; M-1 to K-660; M-1 to C-659; M-1 to D-658; M-1 to R-657; M-1 toH-656; M-1 to F-655; M-1 to I-654; M-1 to N-653; M-1 to I-652; M-1 toK-651; M-1 to S-650; M-1 to P-649; M-1 to E-648; M-1 to F-647; M-1 toH-646; M-1 to L-645; M-1 to S-644; M-1 to A-643; M-1 to N-642; M-1 toI-641; M-1 to Q-640; M-1 to V-639; M-1 to V-638; M-1 to P-637; M-1 toR-636; M-1 to S-635; M-1 to W-634; M-1 to L-633; M-1 to I-632; M-1 toV-631; M-1 to A-630; M-1 to N-629; M-1 to G-628; M-1 to L-627; M-1 toA-626; M-1 to G-625; M-1 to V-624; M-1 to A-623; M-1 to L-622; M-1 toD-621; M-1 to I-620; M-1 to L-619; M-1 to G-618; M-1 to D-617; M-1 toE-616; M-1 to N-615; M-1 to L-614; M-1 to D-613; M-1 to L-612; M-1 toQ-611; M-1 to G-610; M-1 to H-609; M-1 to I-608; M-1 to S-607; M-1 toC-606; M-1 to G-605; M-1 to F-604; M-1 to Y-603; M-1 to Q-602; M-1 toL-601; M-1 to G-600; M-1 to T-599; M-1 to A-598; M-1 to L-597; M-1 toE-596; M-1 to S-595; M-1 to A-594; M-1 to T-593; M-1 to I-592; M-1 toR-591; M-1 to Q-590; M-1 to K-589; M-1 to P-588; M-1 to T-587; M-1 toK-586; M-1 to L-585; M-1 to I-584; M-1 to S-583; M-1 to G-582; M-1 toR-581; M-1 to F-580; M-1 to G-579; M-1 to H-578; M-1 to F-577; M-1 toI-576; M-1 to Y-575; M-1 to I-574; M-1 to A-573; M-1 to G-572; M-1 toA-571; M-1 to H-570; M-1 to N-569; M-1 to D-568; M-1 to E-567; M-1 toL-566; M-1 to P-565; M-1 to A-564; M-1 to G-563; M-1 to V-562; M-1 toV-561; M-1 to V-560; M-1 to D-559; M-1 to N-558; M-1 to Y-557; M-1 toS-556; M-1 to D-555; M-1 to Q-554; M-1 to N-553; M-1 to L-552; M-1 toD-551; M-1 to R-550; M-1 to V-549; M-1 to S-548; M-1 to A-547; M-1 toI-546; M-1 to S-545; M-1 to S-544; M-1 to G-543; M-1 to F-542; M-1 toR-541; M-1 to A-540; M-1 to N-539; M-1 to Q-538; M-1 to Y-537; M-1 toS-536; M-1 to H-535; M-1 to S-534; M-1 to D-533; M-1 to K-532; M-1 toL-531; M-1 to T-530; M-1 to G-529; M-1 to N-528; M-1 to Y-527; M-1 toV-526; M-1 to F-525; M-1 to R-524; M-1 to N-523; M-1 to Q-522; M-1 toR-521; M-1 to L-520; M-1 to E-519; M-1 to Y-518; M-1 to V-517; M-1 toY-516; M-1 to V-515; M-1 to K-514; M-1 to G-513; M-1 to R-512; M-1 toE-511; M-1 to R-510; M-1 to G-509; M-1 to E-508; M-1 to N-507; M-1 toF-506; M-1 to Y-505; M-1 to M-504; M-1 to P-503; M-1 to A-502; M-1 toG-501; M-1 to V-500; M-1 to L-499; M-1 to L-498; M-1 to V-497; M-1 toD-496; M-1 to T-495; M-1 to V-494; M-1 to G-493; M-1 to D-492; M-1 toG-491; M-1 to D-490; M-1 to I-489; M-1 to D-488; M-1 to V-487; M-1 toS-486; M-1 to T-485; M-1 to I-484; M-1 to E-483; M-1 to S-482; M-1 toG-481; M-1 to F-480; M-1 to Y-479; M-1 to S-478; M-1 to G-477; M-1 toI-476; M-1 to Q-474; M-1 to G-474; M-1 to G-473; M-1 to R-472; M-1 toM-471; M-1 to A-470; M-1 to Q-469; M-1 to H-468; M-1 to I-467; M-1 toT-466; M-1 to L-465; M-1 to S-464; M-1 to R-463; M-1 to N-462; M-1 toN-461; M-1 to H-460; M-1 to M-459; M-1 to T-458; M-1 to F-457; M-1 toL-456; M-1 to I-455; M-1 to V-454; M-1 to K-453; M-1 to G-452; M-1 toT-451; M-1 to H-450; M-1 to N-449; M-1 to F-448; M-1 to R-447; M-1 toP-446; M-1 to A-445; M-1 to G-444; M-1 to A-443; M-1 to V-442; M-1 toY-441; M-1 to V-440; M-1 to R-439; M-1 to G-438; M-1 to Q-437; M-1 toR-436; M-1 to S-435; M-1 to S-434; M-1 to V-433; M-1 to V-432; M-1 toS-431; M-1 to T-430; M-1 to V-429; M-1 to T-428; M-1 to Y-427; M-1 toG-426; M-1 to L-425; M-1 to Y-424; M-1 to A-423; M-1 to G-422; M-1 toH-421; M-1 to N-420; M-1 to K-419; M-1 to L-418; M-1 to E-417; M-1 toE-416; M-1 to P-415; M-1 to F-414; M-1 to E-413; M-1 to K-412; M-1 toL-411; M-1 to Y-410; M-1 to S-409; M-1 to E-408; M-1 to R-407; M-1 toL-406; M-1 to P-405; M-1 to I-404; M-1 to V-403; M-1 to K-402; M-1 toG-401; M-1 to A-400; M-1 to S-399; M-1 to T-398; M-1 to E-397; M-1 toK-396; M-1 to L-395; M-1 to V-394; M-1 to A-393; M-1 to G-392; M-1 toN-391; M-1 to W-390; M-1 to D-389; M-1 to Y-388; M-1 to A-387; M-1 toG-386; M-1 to V-385; M-1 to A-384; M-1 to G-383; M-1 to L-382; M-1 toL-381; M-1 to V-380; M-1 to G-379; M-1 to D-378; M-1 to E-377; M-1 toV-376; M-1 to V-375; M-1 to H-374; M-1 to S-373; M-1 to S-372; M-1 toF-371; M-1 to G-370; M-1 to T-369; M-1 to Q-368; M-1 to S-367; M-1 toM-366; M-1 to E-365; M-1 to L-364; M-1 to G-363; M-1 to F-362; M-1 toS-361; M-1 to T-360; M-1 to E-359; M-1 to N-358; M-1 to K-357; M-1 toN-356; M-1 to T-355; M-1 to G-354; M-1 to E-353; M-1 to L-352; M-1 toS-351; M-1 to F-350; M-1 to I-349; M-1 to R-348; M-1 to D-347; M-1 toG-346; M-1 to L-345; M-1 to A-344; M-1 to D-343; M-1 to V-342; M-1 toI-341; M-1 to D-340; M-1 to K-339; M-1 to L-338; M-1 to A-337; M-1 toA-336; M-1 to E-335; M-1 to D-334; M-1 to T-333; M-1 to V-332; M-1 toN-331; M-1 to F-330; M-1 to F-329; M-1 to H-328; M-1 to K-327; M-1 toD-326; M-1 to D-325; M-1 to P-324; M-1 to D-323; M-1 to S-322; M-1 toA-321; M-1 to I-320; M-1 to Y-319; M-1 to K-318; M-1 to I-317; M-1 toE-316; M-1 to N-315; M-1 to L-314; M-1 to F-313; M-1 to T-312; M-1 toE-311; M-1 to P-310; M-1 to N-309; M-1 to I-308; M-1 to G-307; M-1 toR-306; M-1 to R-305; M-1 to N-304; M-1 to Y-303; M-1 to Y-302; M-1 toG-301; M-1 to L-300; M-1 to V-299; M-1 to A-298; M-1 to V-297; M-1 toA-296; M-1 to Y-295; M-1 to R-294; M-1 to T-293; M-1 to V-292; M-1 toN-291; M-1 to D-290; M-1 to R-289; M-1 to E-288; M-1 to S-287; M-1 toQ-286; M-1 to Q-285; M-1 to I-284; M-1 to V-283; M-1 to K-282; M-1 toE-281; M-1 to L-280; M-1 to D-279; M-1 to P-278; M-1 to S-277; M-1 toD-276; M-1 to H-275; M-1 to S-274; M-1 to E-273; M-1 to G-272; M-1 toD-271; M-1 to T-270; M-1 to I-269; M-1 to V-268; M-1 to I-267; M-1 toM-266; M-1 to V-265; M-1 to K-264; M-1 to K-263; M-1 to A-262; M-1 toG-261; M-1 to K-260; M-1 to R-259; M-1 to G-258; M-1 to G-257; M-1 toK-256; M-1 to Q-255; M-1 to F-254; M-1 to A-253; M-1 to E-252; M-1 toS-251; M-1 to R-250; M-1 to A-249; M-1 to F-248; M-1 to E-247; M-1 toI-246; M-1 to G-245; M-1 to F-244; M-1 to A-243; M-1 to T-242; M-1 toR-241; M-1 to T-240; M-1 to E-239; M-1 to T-238; M-1 to G-237; M-1 toG-236; M-1 to R-235; M-1 to Q-234; M-1 to E-233; M-1 to I-232; M-1 toH-231; M-1 to S-230; M-1 to A-229; M-1 to A-228; M-1 to E-227; M-1 toV-226; M-1 to V-225; M-1 to D-224; M-1 to K-223; M-1 to V-222; M-1 toS-221; M-1 to R-220; M-1 to Y-219; M-1 to D-218; M-1 to N-217; M-1 toL-216; M-1 to H-215; M-1 to F-214; M-1 to E-213; M-1 to H-212; M-1 toV-211; M-1 to V-210; M-1 to D-209; M-1 to E-208; M-1 to G-207; M-1 toY-206; M-1 to Q-205; M-1 to V-204; M-1 to V-203; M-1 to G-202; M-1 toV-201; M-1 to Q-200; M-1 to I-199; M-1 to Q-198; M-1 to G-197; M-1 toP-196; M-1 to G-195; M-1 to I-194; M-1 to Y-193; M-1 to F-192; M-1 toK-191; M-1 to K-190; M-1 to L-189; M-1 to I-188; M-1 to N-187; M-1 toI-186; M-1 to L-185; M-1 to F-184; M-1 to H-183; M-1 to Q-182; M-1 toV-181; M-1 to E-180; M-1 to V-179; M-1 to W-178; M-1 to P-177; M-1 toY-176; M-1 to I-175; M-1 to S-174; M-1 to N-173; M-1 to S-172; M-1 toG-171; M-1 to D-170; M-1 to L-169; M-1 to V-168; M-1 to I-167; M-1 toV-166; M-1 to I-165; M-1 to 164; M-1 to M-163; M-1 to Y-162; M-1 toT-161; M-1 to Q-160; M-1 to C-159; M-1 to R-158; M-1 to Q-157; M-1 toL-156; M-1 to A-155; M-1 to P-154; M-1 to A-153; M-1 to V-152; M-1 toT-151; M-1 to K-150; M-1 to S-149; M-1 to F-148; M-1 to R-147; M-1 toF-146; M-1 to N-145; M-1 to S-144; M-1 to N-143; M-1 to V-142; M-1 toR-141; M-1 to S-140; M-1 to C-139; M-1 to M-138; M-1 to G-137; M-1 toT-136; M-1 to T-135; M-1 to Y-134; M-1 to Y-133; M-1 to S-132; M-1 toS-131; M-1 to G-130; M-1 to C-129; M-1 to E-128; M-1 to H-127; M-1 toS-126; M-1 to W-125; M-1 to L-124; M-1 to P-123; M-1 to S-122; M-1 toC-121; M-1 to A-120; M-1 to L-119; M-1 to F-118; M-1 to S-117; M-1 toN-116; M-1 to D-115; M-1 to K-114; M-1 to P-113; M-1 to N-112; M-1 toT-111; M-1 to A-110; M-1 to L-109; M-1 to S-108; M-1 to L-107; M-1 toG-106; M-1 to L-105; M-1 to R-104; M-1 to M-103; M-1 to N-102; M-1 toD-101; M-1 to K-100; M-1 to R-99; M-1 to E-98; M-1 to S-97; M-1 to V-96;M-1 to N-95; M-1 to S-94; M-1 to L-93; M-1 to T-92; M-1 to V-91; M-1 toR-90; M-1 to G-89; M-1 to L-88; M-1 to N-87; M-1 to K-86; M-1 to K-85;M-1 to T-84; M-1 to C-83; M-1 to N-82; M-1 to G-81; M-1 to H-80; M-1 toI-79; M-1 to V-78; M-1 to P-77; M-1 to C-76; M-1 to K-75; M-1 to Y-74;M-1 to V-73; M-1 to D-72; M-1 to G-71; M-1 to T-70; M-1 to K-69; M-1 toQ-68; M-1 to Y-67; M-1 to G-66; M-1 to N-65; M-1 to T-64; M-1 to E-63;M-1 to L-62; M-1 to P-61; M-1 to A-60; M-1 to G-59; M-1 to V-58; M-1 toV-57; M-1 to L-56; M-1 to W-55; M-1 to K-54; M-1 to N-53; M-1 to G-52;M-1 to S-51; M-1 to I-50; M-1 to D-49; M-1 to H-48; M-1 to Q-47; M-1 toQ-46; M-1 to V-45; M-1 to T-44; M-1 to Y-43; M-1 to G-42; M-1 to F-41;M-1 to F-40; M-1 to A-39; M-1 to T-38; M-1 to R-37; M-1 to S-36; M-1 toG-35; M-1 to P-34; M-1 to I-33; M-1 to V-32; M-1 to R-31; M-1 to P-30;M-1 to K-29; M-1 to R-28; M-1 to T-27; M-1 to D-26; M-1 to M-25; M-1 toN-24; M-1 to F-23; M-1 to T-22; M-1 to D-21; M-1 to T-20; M-1 to F-19;M-1 to G-18; M-1 to P-17; M-1 to W-16; M-1 to L-15; M-1 to S-14; M-1 toL-13; M-1 to A-12; M-1 to W-11; M-1 to A-10; M-1 to V-9; M-1 to V-8; M-1to L-7; M-1 to G-6; of SEQ ID NO:35. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g., fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:17 whichhave been determined from the following related cDNA genes: HEEAB54R(SEQ ID NO:104), HRDAF83R (SEQ ID NO:105), HOUBC62R (SEQ ID NO:106),HCDBI19R (SEQ ID NO:107), HOHCU94R (SEQ ID NO:108), HOACC13R (SEQ IDNO:109), HCDAP21R (SEQ ID NO:110), HNHHA34R (SEQ ID NO:111), HOHEA75R(SEQ ID NO:112) and HNGEL59R (SEQ ID NO:113).

A polynucleotide encoding a polypeptide of the present invention isobtained from human ovary small intestine, fetal heart, fetal brain,large intestine, osteoblasts, human trabelcular bone cells, messangialcells, adipocytes, osteosarcoma, chondrosarcoma, breast cancer cells,and bone marrow tissues and cells. The polynucleotide of this inventionwas discovered in a human osteoblast n cDNA library.

Based on the sequence similarity to the human integrin alpha 1 subunit,translation product of this gene is expected to share at least somebiological activities with integrin proteins, and specifically theintegrin alpha 1 protein. Such activities are known in the art, some ofwhich are described elsewhere herein.

Specifically, polynucleotides and polypeptides of the invention,including antibodies, are also useful for modulating the differentiationof normal and malignant cells, modulating the proliferation and/ordifferentiation of cancer and neoplastic cells, and modulating theimmune response. Polynucleotides and polypeptides of the invention mayrepresent a diagnostic marker for hematopoietic and immune diseasesand/or disorders. The full-length protein should be a secreted protein,based upon homology to the integrin family. Therefore, it is secretedinto serum, urine, or feces and thus the levels is assayable frompatient samples. Assuming specific expression levels are reflective ofthe presence of immune disorders, this protein would provide aconvenient diagnostic for early detection. In addition, expression ofthis gene product may also be linked to the progression of immunediseases, and therefore may itself actually represent a therapeutic ortherapeutic target for the treatment of cancer.

Polynucleotides and polypeptides of the invention may play an importantrole in the pathogenesis of human cancers and cellular transformation,particularly those of the immune and hematopoietic systems.Polynucleotides and polypeptides of the invention may also be involvedin the pathogenesis of developmental abnormalities based upon itspotential effects on proliferation and differentiation of cells andtissue cell types. Due to the potential proliferating anddifferentiating activity of said polynucleotides and polypeptides, theinvention is useful as a therapeutic agent in inducing tissueregeneration, for treating inflammatory conditions (e.g., inflammatorybowel syndrome, diverticulitis, etc.). Moreover, the invention is usefulin modulating the immune response to aberrant polypeptides, as may existin rapidly proliferating cells and tissue cell types, particularly inadenocarcinoma cells, and other cancers.

Alternatively, the expression within cellular sources marked byproliferating cells indicates this protein may play a role in theregulation of cellular division, and may show utility in the diagnosis,treatment, and/or prevention of developmental diseases and disorders,including cancer, and other proliferative conditions. Representativeuses are described in the “Hyperproliferative Disorders” and“Regeneration” sections below and elsewhere herein. Briefly,developmental tissues rely on decisions involving cell differentiationand/or apoptosis in pattern formation.

Dysregulation of apoptosis can result in inappropriate suppression ofcell death, as occurs in the development of some cancers, or in failureto control the extent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA).

Alternatively, this gene product is involved in the pattern of cellularproliferation that accompanies early embryogenesis. Thus, aberrantexpression of this gene product in tissues—particularly adulttissues—may correlate with patterns of abnormal cellular proliferation,such as found in various cancers. Because of potential roles inproliferation and differentiation, this gene product may haveapplications in the adult for tissue regeneration and the treatment ofcancers. It may also act as a morphogen to control cell and tissue typespecification. Therefore, the polynucleotides and polypeptides of thepresent invention are useful in treating, detecting, and/or preventingsaid disorders and conditions, in addition to other types ofdegenerative conditions. Thus, this protein may modulate apoptosis ortissue differentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases. Theprotein is useful in modulating the immune response to aberrantpolypeptides, as may exist in proliferating and cancerous cells andtissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

This gene is expressed almost exclusively in osteoblasts, humantrabelcular bone cells, messangial cells, adipocytes, and to a lesserextent in osteosarcoma, chondrosarcoma, breast cancer cells, and bonemarrow.

Therefore, polynucleotides and polypeptides of the invention, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of the following diseases and conditions which include, butare not limited to, disorders of the skeletal system, connectivetissues, and immune and hematpoietic diseases and/or disorders.Similarly, polypeptides and antibodies directed to these polypeptidesare useful to provide immunological probes for differentialidentification of the tissue(s) or cell type(s). For a number ofdisorders of the above tissues or cells, particularly of the connectivetissue and skeletal system, expression of this gene at significantlyhigher or lower levels is detected in certain tissues or cell types(e.g. immune, hematopoietic, skeletal, bone, cartilage, developmental,reproductive, secretory, and cancerous and wounded tissues) or bodilyfluids or cell types (e.g., lymph, serum, plasma, urine, synovial fluidor spinal fluid) or another tissue or cell sample taken from anindividual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue from anindividual not having the disorder.

The tissue distribution in osteoblasts and homology to integrin alphasubunit 10 indicates that the protein products of this gene are usefulfor the treatment of disorders and conditions affecting the skeletalsystem, in particular osteoporosis as well as disorders afflictingconnective tissues (e.g., arthritis, trauma, tendonitis, chrondomalaciaand inflammation), such as in the diagnosis and treatment of variousautoimmune disorders such as rheumatoid arthritis, lupus, scleroderma,and dermatomyositis as well as dwarfism, spinal deformation, andspecific joint abnormalities as well as chondrodysplasias (i.e.,spondyloepiphyseal dysplasia congenita, familial osteoarthritis,Atelosteogenesis type II, metaphyseal chondrodysplasia type Schmid).polynucleotides and polypeptides corresponding to this gene are usefulfor the treatment and diagnosis of hematopoietic related disorders suchas anemia, pancytopenia, leukopenia, thrombocytopenia or leukemia sincestromal cells are important in the production of cells of hematopoieticlineages. Such a use is consistent with the observed homology tointegrin family members, in conjunction with the tissue distribution inbone marrow cells.

Integrins play pivotal roles in cell migration, inflammation,proliferation, and cellular infiltration. Thus, the present invention isexpected to share at least some of these activities. Representative usesare described in the “Immune Activity” and “infectious disease” sectionsbelow, in Example 11, 13, 14, 16, 18, 19, 20, and 27, and elsewhereherein. Briefly, the uses include bone marrow cell ex-vivo culture, bonemarrow transplantation, bone marrow reconstitution, radiotherapy orchemotherapy of neoplasia. The gene product may also be involved inlymphopoiesis, therefore, it can be used in immune disorders such asinfection, inflammation, allergy, immunodeficiency etc. In addition,this gene product may have commercial utility in the expansion of stemcells and committed progenitors of various blood lineages, and in thedifferentiation and/or proliferation of various cell types. Based uponthe tissue distribution of this protein, antagonists directed againstthis protein is useful in blocking the activity of this protein.Accordingly, preferred are antibodies which specifically bind a portionof the translation product of this gene.

Also provided is a kit for detecting tumors in which expression of thisprotein occurs. Such a kit comprises in one embodiment an antibodyspecific for the translation product of this gene bound to a solidsupport. Also provided is a method of detecting these tumors in anindividual which comprises a step of contacting an antibody specific forthe translation product of this gene to a bodily fluid from theindividual, preferably serum, and ascertaining whether antibody binds toan antigen found in the bodily fluid. Preferably the antibody is boundto a solid support and the bodily fluid is serum. The above embodiments,as well as other treatments and diagnostic tests (kits and methods), aremore particularly described elsewhere herein.

Furthermore, the protein may also be used to determine biologicalactivity, to raise antibodies, as tissue markers, to isolate cognateligands or receptors, to identify agents that modulate theirinteractions, in addition to its use as a nutritional supplement.Protein, as well as, antibodies directed against the protein may showutility as a tumor marker and/or immunotherapy targets for the abovelisted tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:17 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 4981 of SEQID NO:17, b is an integer of 15 to 4995, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:17, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 8

The present invention relates to a novel peptidoglycan recognitionbinding proteins expressed by chondrosarcoma tissue. More specifically,isolated nucleic acid molecules are provided encoding a humanpeptidoglycan recognition protein-related protein, sometimes referred toherein as “human tag7” or “tag7” or “htag7”. Further provided arevectors, host cells and recombinant methods for producing the same. Theinvention also relates to both the inhibition and enhancement ofactivities of the tag7 protein, polypeptides and diagnostic methods fordetecting tag7 gene expression.

Peptidoglycan, as well as Lipopolysaccharide (LPS), is a surfacecomponent of many bacteria which illicit a wide range of physiologicaland immune responses in humans. Specifically, peptidoglycan has beenshown to manifest itself clinically by reproducing most of the symptomsof bacterial infection, including fever, acute-phase response,inflammation, septic shock, leukocytosis, sleepiness, malaise, abcessformation, and arthritis (see Dziarski et al., JBC, 273 (15):8680(1998)). Furthermore, the type of peptidoglycan (i.e.—the specificstereoisomers or analogs of muramyl dipeptide,N-acetylglucosaminyl-beta(1-4)-N-acteylmuramyl tetrapeptides, etc.),were shown to elicit a broad range of activities, including exhibitinggreater pyrogenicity, inducing acute joint inflammation, stimulatingmacrophages, and causing hemorrhagic necrosis at a primed site (SeeKotani et al., Fed Proc, 45(11):2534 (1986)). It has been demonstratedin humans that a lipopolysaccharide binding protein exists that wasdiscovered as a trace plasma protein (See Schumann et al., Science,249(4975):1429 (1990)). It is thought that one of the modes of action bywhich this lipopolysaccharide binding protein functions is by forminghigh-affinity complexes with lipopolysaccharide, that then bind tomacrophages and monocytes, inducing the secretion of tumor necrosisfactor. Dziarski and Gupta (See Dziarski et al., JBC, 269(3):2100(1994)) demonstrated that a 70 kDa receptor protein present on thesurface of mouse lymphocytes served to bind heparin, heparinoids,bacterial lipoteichoic acids, peptidoglycan, and lipopolysaccharides.Recently, Dziarski et al. demonstrated that the CD14, aglycosylphosphatidylinositol-linked protein present on the surface ofmacrophage and polymorphonuclear leukocytes, bound peptidoglycan andlipopolysaccharide.

Furthermore, the binding affinity of CD14 for lipopolysaccharide wassignificantly increased in the presence of a LPS-binding protein presentin plasma. It is thought that the LPS-binding protein functions as atransfer molecule, whereby it binds LPS and presents it to the CD14receptor (See Dziarski et al., JBC, 273(15):8680 (1998)). Yoshida et al.isolated a peptidoglycan binding protein from the hemolymph of theSilkworm, Bombyx mori, using column chromatography. This protein wasfound to have a very specific affinity for peptidoglycan (See Yoshida etal., JBC, 271(23):13854 (1996)).

Additionally, Kang et al. recently cloned a peptidoglycan bindingprotein from the moth Trichoplusia ni. The peptidoglycan binding proteinwas shown to bind strongly to insoluble peptidoglycan (See Kang et al.,PNAS, 95(17):10078 (1998)). In this study the peptidoglycan bindingprotein was upregulated by a bacterial infection in T. ni. The insectimmune system is regarded as a model for innate immunity. Thus, Kang etal were able to gene both mouse and human homologs of the T. nipeptidoglycan binding protein. All of these peptidoglycan bindingproteins shared regions of homology, as well as four conserved cysteineresidues which may function in the tertiary structure of the protein,possibly in helping to form binding domains. Given that peptidoglycan isan integral component of bacterial cell walls, and that it induces manyphysiological responses from cytokine secretion to inflammation andmacrophage activation, it appears as if this family of proteins is aubiquitous group involved in the binding and recognition ofpeptidoglycan, the presentation of antigens (e.g., cell wall components,etc.), and the activation of the immune system, such as the secretion ofcytokines, such as TNF. TNF is noted for its pro-inflammatory actionswhich result in tissue injury, such as induction of procoagulantactivity on vascular endothelial cells (Pober, J. S. et al., J. Immunol.136:1680 (1986)), increased adherence of neutrophils and lymphocytes(Pober, J. S. et al., J. Immunol. 138:3319 (1987)), and stimulation ofthe release of platelet activating factor from macrophages, neutrophilsand vascular endothelial cells (Camussi, G. et al., J. Exp. Med.166:1390 (1987)).

Recent evidence implicates TNF in the pathogenesis of many infections(Cerami, A. et al., Immunol. Today 9:28 (1988)), immune disorders,neoplastic pathology, e.g., in cachexia accompanying some malignancies(Oliff, A. et al., Cell 50:555 (1987)), and in autoimmune pathologiesand graft-versus host pathology (Piguet, P.-F. et al., J. Exp. Med.166:1280 (1987)). The association of TNF with cancer and infectiouspathologies is often related to the host's catabolic state. A majorproblem in cancer patients is weight loss, usually associated withanorexia. The extensive wasting which results is known as “cachexia”(Kem, K. A. et al. J. Parent. Enter. Nutr. 12:286-298 (1988)). Cachexiaincludes progressive weight loss, anorexia, and persistent erosion ofbody mass in response to a malignant growth. The cachectic state is thusassociated with significant morbidity and is responsible for themajority of cancer mortality.

A number of studies have suggested that TNF is an important mediator ofthe cachexia in cancer, infectious pathology, and in other catabolicstates. TNF is thought to play a central role in the pathophysiologicalconsequences of Gram-negative sepsis and endotoxic shock (Michie, H. R.et al., Br. J. Surg. 76:670-671 (1989); Debets, J. M. H. et al., SecondVienna Shock Forum, p. 463-466 (1989); Simpson, S. Q. et al., Crit. CareClin. 5:27-47 (1989)), including fever, malaise, anorexia, and cachexia.Endotoxin is a potent monocyte/macrophage activator which stimulatesproduction and secretion of TNF (Kombluth, S. K. et al., J. Immunol.137:2585-2591 (1986)) and other cytokines. Because TNF could mimic manybiological effects of endotoxin, it was concluded to be a centralmediator responsible for the clinical manifestations ofendotoxin-related illness. TNF and other monocyte-derived cytokinesmediate the metabolic and neurohormonal responses to endotoxin (Michie,H. R. et al., N. Eng. J. Med. 318:1481-1486 (1988)). Endotoxinadministration to human volunteers produces acute illness with flu-likesymptoms including fever, tachycardia, increased metabolic rate andstress hormone release (Revhaug, A. et al., Arch. Surg. 123:162-170(1988)). Elevated levels of circulating TNF have also been found inpatients suffering from Gram-negative sepsis (Waage, A. et al., Lancet1:355-357 (1987); Hammerle, A. F. et al., Second Vienna Shock Forum p.715-718 (1989); Debets, J. M. H. et al., Crit. Care Med. 17:489-497(1989); Calandra, T. et al., J. Infec. Dis. 161:982-987 (1990)). Passiveimmunotherapy directed at neutralizing TNF may have a beneficial effectin Gram-negative sepsis and endotoxemia, based on the increased TNFproduction and elevated TNF levels in these pathology states, asdiscussed above.

Antibodies to a “modulator” material which was characterized ascachectin (later found to be identical to TNF) were disclosed by Ceramiet al. (EPO Patent Publication 0,212,489, Mar. 4, 1987). Such antibodieswere said to be useful in diagnostic immunoassays and in therapy ofshock in bacterial infections. Rubin et al. (EPO Patent Publication0,218,868, Apr. 22, 1987) disclosed monoclonal antibodies to human TNF,the hybridomas secreting such antibodies, methods of producing suchantibodies, and the use of such antibodies in immunoassay of TNF. Yoneet al. (EPO Patent Publication 0,288,088, Oct. 26, 1988) disclosedanti-TNF antibodies, including mAbs, and their utility in immunoassaydiagnosis of pathologies, in particular Kawasaki's pathology andbacterial infection. The body fluids of patients with Kawasaki'spathology (infantile acute febrile mucocutaneous lymph node syndrome;Kawasaki, T., Allergy 16:178 (1967); Kawasaki, T., Shonica (Pediatrics)26:935 (1985)) were said to contain elevated TNF levels which wererelated to progress of the pathology (Yone et al., supra).

Accordingly, there is a need to provide molecules that are involved inpathological conditions. Such novel proteins could be useful inaugmenting the immune system in such areas as immune recognition,antigen presentation, and immune system activation. Antibodies orantagonists directed against these proteins is useful in reducing oreliminating disorders associated with TNF and TNF-like cytokines, suchas endotoxic shock and auto-immune disorders, for example.

The polypeptide of the present invention has been putatively identifiedas a member of the novel peptidoglycan recognition binding proteinfamily and has been termed human tag7. This identification has been madeas a result of amino acid sequence homology to the mouse tag7 (SeeGenbank Accession No. emb|CAA60133).

The translation product of this gene also shares sequence homology withantimicrobial BGP-A, a bovine antimicrobial peptide from bovineneutrophils. Preferred polypeptides of this invention comprise residues184 to 196 shown in SEQ ID NO: 36. This polypeptide is believed to bethe active mature form of the translation product of this gene.

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, or all five ofthe immunogenic epitopes shown in SEQ ID NO: 36 as residues: Ala-63 toAsn-68, Ala-71 to Gln-81, Tyr-135 to Thr-141, Leu-167 to Gln-174, and/orPro-191 to Pro-196. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

FIG. 34 shows the nucleotide (SEQ ID NO:18) and deduced amino acidsequence (SEQ ID NO:36) of htag7. Predicted amino acids from about 1 toabout 21 constitute the predicted signal peptide (amino acid residuesfrom about 1 to about 21 in SEQ ID NO:36) and are represented by theunderlined amino acid regions; and amino acids from about 34 to about117 constitute the predicted PGRP-like domain (amino acids from about 34to about 117 in SEQ ID NO:36) and are represented by the doubleunderlined amino acids.

FIG. 35 shows the regions of similarity between the amino acid sequencesof the htag7 protein (SEQ ID NO:36) and the mouse tag7 protein (SEQ IDNO:114).

FIG. 36 shows an analysis of the htag7 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown.

As shown in FIG. 34, htag7 has a PGRP domain (the PGRP domain compriseamino acids from about 34 to about 117 of SEQ ID NO:36; which correspondto amino acids from about 34 to about 117 of FIG. 34). Thepolynucleotide contains an open reading frame encoding the htag7polypeptide of 198 amino acids. htag7 exhibits a high degree of homologyat the amino acid level to the mouse tag7 (as shown in FIG. 35). Thepresent invention provides isolated nucleic acid molecules comprising apolynucleotide encoding the htag7 polypeptide having the amino acidsequence shown in FIG. 34 (SEQ ID NO:36). The nucleotide sequence shownin FIG. 34 (SEQ ID NO:18) was obtained by sequencing a cloned cDNA(HCDDP40), which was deposited on November 17 at the American TypeCulture Collection, and given Accession Number 203484.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO:18 is intended DNA fragments atleast about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:18. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:18. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Representative examples of htag7 polynucleotide fragments ofthe invention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, from about 501 toabout 550, from about 551 to about 600, from about 601 to about 650,from about 651 to about 700, from about 701 to about 726, and from about130 to about 379 of SEQ ID NO:18, or the complementary strand thereto,or the cDNA contained in the deposited gene. In this context “about”includes the particularly recited ranges, larger or smaller by several(5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding a member selected from the group: apolypeptide comprising or alternatively, consisting of, the PGRP-likedomain (amino acid residues from about 34 to about 117 in FIG. 34 (aminoacids from about 34 to about 117 in SEQ ID NO:36). Since the location ofthese domains have been predicted by computer analysis, one of ordinaryskill would appreciate that the amino acid residues constituting thesedomains may vary slightly (e.g., by about 1 to 15 amino acid residues)depending on the criteria used to define each domain. As indicated,nucleic acid molecules of the present invention which encode a htag7polypeptide may include, but are not limited to those encoding the aminoacid sequence of the PGRP-like domain of the polypeptide, by itself, andthe coding sequence for the PGRP-like domain of the polypeptide andadditional sequences, such as a pre-, or pro or prepro-protein sequence.In additional embodiments, the polynucleotides of the invention encodefunctional attributes of htag7.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of htag7. The data representing the structural orfunctional attributes of htag7 set forth in FIG. 36 and/or Table XII, asdescribed above, was generated using the various modules and algorithmsof the DNA*STAR set on default parameters. In a preferred embodiment,the data presented in columns VIII, IX, XIII, and XIV of Table XII canbe used to determine regions of htag7 which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX, XIII, and/or XIV bychoosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

Certain preferred regions in these regards are set out in FIG. 36, butmay, as shown in Table XII, be represented or identified by usingtabular representations of the data presented in FIG. 36. The DNA*STARcomputer algorithm used to generate FIG. 36 (set on the original defaultparameters) was used to present the data in FIG. 36 in a tabular format(See Table XII). The tabular format of the data in FIG. 36 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 36 and in Table XIIinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIG. 34. Asset out in FIG. 36 and in Table XII, such preferred regions includeGarnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, modulate cellularinteraction, or signalling pathways, etc.) may still be retained. Forexample, the ability of shortened htag7 muteins to induce and/or bind toantibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that an htag7 mutein with a largenumber of deleted N-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six htag7 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the htag7 aminoacid sequence shown in FIG. 34, up to the proline residue at positionnumber 191 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-196 of FIG. 34 , where n1 is aninteger from 2 to 191 corresponding to the position of the amino acidresidue in FIG. 34 (which is identical to the sequence shown as SEQ IDNO:36). In another embodiment, N-terminal deletions of the htag7polypeptide can be described by the general formula n2-196, where n2 isa number from 2 to 191, corresponding to the position of amino acididentified in FIG. 34. N-terminal deletions of the htag7 polypeptide ofthe invention shown as SEQ ID NO:36 include polypeptides comprising theamino acid sequence of residues: N-terminal deletions of the htag7polypeptide of the invention shown as SEQ ID NO:36 include polypeptidescomprising the amino acid sequence of residues: S-2 to P-196; R-3 toP-196; R-4 to P-196; S-5 to P-196; M-6 to P-196; L-8 to P-196; A-9 toP-196; W-10 to P-196; A-11 to P-196; L-12 to P-196; P-13 to P-196; S-14to P-196; L-15 to P-196; L-16 to P-196; R-17 to P-196; L-18 to P-196;G-19 to P-196; A-20 to P-196; A-21 to P-196; Q-22 to P-196; E-23 toP-196; T-24 to P-196; E-25 to P-196; D-26 to P-196; P-27 to P-196; A-28to P-196; C-29 to P-196; C-30 to P-196; S-31 to P-196; P-32 to P-196;I-33 to P-196; V-34 to P-196; P-35 to P-196; R-36 to P-196; N-37 toP-196; E-38 to P-196; W-39 to P-196; K-40 to P-196; A-41 to P-196; L-42to P-196; A-43 to P-196; S-44 to P-196; E-45 to P-196; C-46 to P-196;A-47 to P-196; Q-48 to P-196; H-49 to P-196; L-50 to P-196; S-51 toP-196; L-52 to P-196; P-53 to P-196; L-54 to P-196; R-55 to P-196; Y-56to P-196; V-57 to P-196; V-58 to P-196; V-59 to P-196; S-60 to P-196;H-61 to P-196; T-62 to P-196; A-63 to P-196; G-64 to P-196; S-65 toP-196; S-66 to P-196; C-67 to P-196; N-68 to P-196; T-69 to P-196; P-70to P-196; A-71 to P-196; S-72 to P-196; C-73 to P-196; Q-74 to P-196;Q-75 to P-196; Q-76 to P-196; A-77 to P-196; R-78 to P-196; N-79 toP-196; V-80 to P-196; Q-81 to P-196; H-82 to P-196; Y-83 to P-196; H-84to P-196; M-85 to P-196; K-86 to P-196; T-87 to P-196; L-88 to P-196;G-89 to P-196; W-90 to P-196; C-91 to P-196; D-92 to P-196; V-93 toP-196; G-94 to P-196; Y-95 to P-196; N-96 to P-196; F-97 to P-196; L-98to P-196; I-99 to P-196; G-100 to P-196; E-101 to P-196; D-102 to P-196;G-103 to P-196; L-104 to P-196; V-105 to P-196; Y-106 to P-196; E-107 toP-196; G-108 to P-196; R-109 to P-196; G-110 to P-196; W-111 to P-196;N-112 to P-196; F-113 to P-196; T-114 to P-196; G-115 to P-196; A-116 toP-196; H-117 to P-196; S-118 to P-196; G-119 to P-196; H-120 to P-196;L-121 to P-196; W-122 to P-196; N-123 to P-196; P-124 to P-196; M-125 toP-196; S-126 to P-196; I-127 to P-196; G-128 to P-196; I-129 to P-196;S-130 to P-196; F-131 to P-196; M-132 to P-196; G-133 to P-196; N-134 toP-196; Y-135 to P-196; M-136 to P-196; D-137 to P-196; R-138 to P-196;V-139 to P-196; P-140 to P-196; T-141 to P-196; P-142 to P-196; Q-143 toP-196; A-144 to P-196; I-145 to P-196; R-146 to P-196; A-147 to P-196;A-148 to P-196; Q-149 to P-196; G-150 to P-196; L-151 to P-196; L-152 toP-196; A-153 to P-196; C-154 to P-196; G-155 to P-196; V-156 to P-196;A-157 to P-196; Q-158 to P-196; G-159 to P-196; A-160 to P-196; L-161 toP-196; R-162 to P-196; S-163 to P-196; N-164 to P-196; Y-165 to P-196;V-166 to P-196; L-167 to P-196; K-168 to P-196; G-169 to P-196; H-170 toP-196; R-171 to P-196; D-172 to P-196; V-173 to P-196; Q-174 to P-196;R-175 to P-196; T-176 to P-196; L-177 to P-196; S-178 to P-196; P-179 toP-196; G-180 to P-196; N-181 to P-196; Q-182 to P-196; L-183 to P-196;Y-184 to P-196; H-185 to P-196; L-186 to P-196; I-187 to P-196; Q-188 toP-196; N-189 to P-196; W-190 to P-196; P-191 to P-196; of SEQ ID NO:36.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities ) may still be retained. For example theability of the shortened htag7 mutein to induce and/or bind toantibodies which recognize the complete or mature forms of thepolypeptide generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a htag7 mutein with a largenumber of deleted C-terminal amino acid residues may retain somebiological or immunogenic activities. In fact, peptides composed of asfew as six htag7 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the htag7 polypeptide shown in FIG. 34, up to the methionineresidue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-ml of FIG. 1, where mlis an integer from 6 to 196 corresponding to the position of the aminoacid residue in FIG. 34. Moreover, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of C-terminal deletions of thehtag7 polypeptide of the invention shown as SEQ ID NO:36 includepolypeptides comprising the amino acid sequence of residues: M-1 toS-195; M-1 to R-194; M-1 to Y-193; M-1 to H-192; M-1 to P-191; M-1 toW-190; M-1 to N-189; M-1 to Q-188; M-1 to I-187; M-1 to L-186; M-1 toH-185; M-1 to Y-184; M-1 to L-183; M-1 to Q-182; M-1 to N-181; M-1 toG-180; M-1 to P-179; M-1 to S-178; M-1 to L-177; M-1 to T-176; M-1 toR-175; M-1 to Q-174; M-1 to V-173; M-1 to D-172; M-1 to R-171; M-1 toH-170; M-1 to G-169; M-1 to K-168; M-1 to L-167; M-1 to V-166; M-1 toY-165; M-1 to N-164; M-1 to S-163; M-1 to R-162; M-1 to L-161; M-1 toA-160; M-1 to G-159; M-1 to Q-158; M-1 to A-157; M-1 to V-156; M-1 toG-155; M-1 to C-154; M-1 to A-153; M-1 to L-152; M-1 to L-151; M-1 toG-150; M-1 to Q-149; M-1 to A-148; M-1 to A-147; M-1 to R-146; M-1 toI-145; M-1 to A-144; M-1 to Q-143; M-1 to P-142; M-1 to T-141; M-1 toP-140; M-1 to V-139; M-1 to R-138; M-1 to D-137; M-1 to M-136; M-1 toY-135; M-1 to N-134; M-1 to G-133; M-1 to M-132; M-1 to F-131; M-1 toS-130; M-1 to l-129; M-1 to G-128; M-1 to I-127; M-1 to S-126; M-1 toM-125; M-1 to P-124; M-1 to N-123; M-1 to W-122; M-1 to L-121; M-1 toH-120; M-1 to G-119; M-1 to S-118; M-1 to H-117; M-1 to A-116; M-1 toG-115; M-1 to T-114; M-1 to F-113; M-1 to N-112; M-1 to W-111; M-1 toG-110; M-1 to R-109; M-1 to G-108; M-1 to E-107; M-1 to Y-106; M-1 toV-105; M-1 to L-104; M-1 to G-103; M-1 to D-102; M-1 to E-101; M-1 toG-100; M-1 to I-99; M-1 to L-98; M-1 to F-97; M-1 to N-96; M-1 to Y-95;M-1 to G-94; M-1 to V-93; M-1 to D-92; M-1 to C-91; M-1 to W-90; M-1 toG-89; M-1 to L-88; M-1 to T-87; M-1 to K-86; M-1 to M-85; M-1 to H-84;M-1 to Y-83; M-1 to H-82; M-1 to Q-81; M-1 to V-80; M-1 to N-79; M-1 toR-78; M-1 to A-77; M-1 to Q-76; M-1 to Q-75; M-1 to Q-74; M-1 to C-73;M-1 to S-72; M-1 to A-71; M-1 to P-70; M-1 to T-69; M-1 to N-68; M-1 toC-67; M-1 to S-66; M-1 to S-65; M-1 to G-64; M-1 to A-63; M-1 to T-62;M-1 to H-61; M-1 to S-60; M-1 to V-59; M-1 to V-58; M-1 to V-57; M-1 toY-56; M-1 to R-55; M-1 to L-54; M-1 to P-53; M-1 to L-52; M-1 to S-51;M-1 to L-50; M-1 to H-49; M-1 to Q-48; M-1 to A-47; M-1 to C-46; M-1 toE-45; M-1 to S-44; M-1 to A-43; M-1 to L-42; M-1 to A-41; M-1 to K-40;M-1 to W-39; M-1 to E-38; M-1 to N-37; M-1 to R-36; M-1 to P-35; M-1 toV-34; M-1 to I-33; M-1 to P-32; M-1 to S-31; M-1 to C-30; M-1 to C-29;M-1 to A-28; M-1 to P-27; M-1 to D-26; M-1 to E-25; M-1 to T-24; M-1 toE-23; M-1 to Q-22; M-1 to A-21; M-1 to A-20; M-1 to G-19; M-1 to L-18;M-1 to R-17; M-1 to L-16; M-1 to L-15; M-1 to S-14; M-1 to P-13; M-1 toL-12; M-1 to A-11; M-1 to W-10; M-1 to A-9; M-1 to L-8; M-1 to L-7; M-1to M-6; of SEQ ID NO:36. Polynucleotides encoding these polypeptides arealso encompassed by the invention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:36 whichhave been determined from the following related cDNA genes: HBMTB79R(SEQ ID NO:115) and HCDDP-40R (SEQ ID NO:116).

A polynucleotide encoding a polypeptide of the present invention isobtained from human chondrosarcoma cells, bone marrow, and neutrophils.The polynucleotide of this invention was discovered in a humanchondrosarcoma cDNA library. This gene is expressed primarily in bonemarrow and to a lesser extent in human chondrosarcoma and neutrophils.

Therefore, polynucleotides and polypeptides of the invention, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of diseases and conditions which include, but are not limitedto, infections, cancer, and disorders of the immune system. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of infected tissues and the immunesystem, expression of this gene at significantly higher or lower levelsis routinely detected in certain tissues or cell types (e.g. immune,hematopoietic, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, serum, plasma, urine, synovial fluid and spinal fluid) oranother tissue or cell sample taken from an individual having such adisorder, relative to the standard gene expression level, i.e., theexpression level in healthy tissue or bodily fluid from an individualnot having the disorder.

Based on the sequence similarity to the mouse tag7 and the PGRP-likedomain, translation product of this gene is expected to share at leastsome biological activities with tag7 proteins, and specifically cytokinemodulatory proteins. Such activities are known in the art, some of whichare described elsewhere herein. Specifically, polynucleotides andpolypeptides of the invention are also useful for modulating thedifferentiation of normal and malignant cells, modulating theproliferation and/or differentiation of cancer and neoplastic cells,such as chondrosarcomas, and modulating the immune response.Polynucleotides and polypeptides of the invention may represent adiagnostic marker for hematopoietic and immune diseases and/ordisorders. The full-length protein should be a secreted protein, basedupon homology to the tag7 protein. Therefore, it is secreted into serum,urine, or feces and thus the levels is assayable from patient samples.Assuming specific expression levels are reflective of the presence ofimmune disorders, this protein would provide a convenient diagnostic forearly detection. In addition, expression of this gene product may alsobe linked to the progression of immune diseases, and therefore mayitself actually represent a therapeutic or therapeutic target for thetreatment of cancer.

Polynucleotides and polypeptides of the invention may play an importantrole in the pathogenesis of human cancers and cellular transformation,particularly those of the immune and hematopoietic systems.Polynucleotides and polypeptides of the invention may also be involvedin the pathogenesis of developmental abnormalities based upon itspotential effects on proliferation and differentiation of cells andtissue cell types. Due to the potential proliferating anddifferentiating activity of said polynucleotides and polypeptides, theinvention is useful as a therapeutic agent in inducing tissueregeneration, for treating inflammatory conditions (e.g., inflammatorybowel syndrome, diverticulitis, etc.).

Moreover, the invention is useful in modulating the immune response toaberrant polypeptides, as may exist in rapidly proliferating cells andtissue cell types, particularly in adenocarcinoma cells, and othercancers. The translation product of this gene shares sequence homologywith Tag7, which is a mouse cytokine that, in soluble form, triggersapoptosis in mouse L929 cells in vitro.

Features of Protein Encoded by Gene No: 9

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. The polypeptide of the present invention has beenputatively identified as a human butyrophilin homolog derived from ahuman testes tumor cDNA library. The polypeptide of the presentinvention is sometimes hereafter referred to as “Butyrophlin and B7-likeIgG superfamily receptor”, and/or “BBIR II”. The invention also relatesto inhibiting the action of such polypeptides.

Butyrophilin is a glycoprotein of the immunoglobulin superfamily that issecreted in association with the milk-fat-globule membrane from mammaryepithelial cells. The butyrophilin gene appears to have evolved from asubset of genes in the immunoglobulin superfamily and genes encoding theB30.2 domain, which is conserved in a family of zinc-finger proteins.Furthermore, expression analysis of butyrophilin genes has shown thatbutyrophilin expression increases during lactation in conjunction withan increase in milk fat content. These results suggest that thestage-specific expression of milk fat globule membrane glycoproteins inmammary epithelial cells is regulated in a similar but not necessarilyidentical mechanism to that of a major milk protein, beta-casein.

The polypeptide of the present invention has been putatively identifiedas a member of the milk fat globule membrane glycoprotein family, andmore particularly the butyrophilin family, and has been termedButyrophlin and B7-like IgG superfamily receptor (“BBIR II”). Thisidentification has been made as a result of amino acid sequence homologyto the bovine butyrophilin precursor (See Genbank Accession No.gi|162773).

Preferred polynucleotides of the invention comprise, or alternativelyconsist of, a nucleic acid sequence selected from the group consistingof:ACATCCATGGCTCTAATGCTCAGTTTGGTTCTGAGTCTCCTCAAGCTGGGATCAGGGCAGTGGCAGGTGTTTGGGCCAGACAAGCCTGTCCAGGCCTTGGTGGGGGAGGACGCAGCATTCTCCTGTTTCCTGTCTCCTAAGACCAATGCAGAGGCCATGGAAGTGCGGTTCTTCAGGGGCCAGTTCTCTAGCGTGGTCCACCTCTACAGGGACGGGAAGGACCAGCCATTTATGCAGATGCCACAGTATCAAGGCAGGACAAAACTGGTGAAGGATTCTATTGCGGAGGGGCGCATCTCTCTGAGGCTGGAAAACATTACTGTGTTGGATGCTGGCCTCTATGGGTGCAGGATTAGTTCCCAGTCTTACTACCAGAAGGCCATCTGGGAGCTACAGGTGTCAGCACTGGGCTCAGTrCCTCTCATTTCCATCACGGGATATGTTGATAGAGACATCCAGCTACTCTGTCAGTCCTCGGGCTGGTTCCCCCGGCCCACAGCGAAGTGGAAAGGTCCACAAGGACAGGATTTGTCCACAGACTCCAGGACAAACAGAGACATGCATGGCCTGTTTGATGTGGAGATCTCTCTGACCGTCCAAGAGAACGCCGGGAGCATATCCTGTTCCATGCGGCATGCTCATCTGAGCCGAGAGGTGGAATCCAGGGTACAGATAGGAGATACCTTTTTCGAGCCTATATCGTGGCACCTGGCTACCAAAGTACTGGGAATACTCTGCTGTGGCCTATTTTTTGGCATTGTTGGACTGAAGATTTrCTTCTCCAAATTCCAGTGGAAAATCCAGGCGGAACTGGACTGGAGAAGAAAGCACGGACAGGCAGAATTGAGAGACGCCCGGAAACACGCAGTGGAGGTGACTCTGGATCCAGAGACGGCTCACCCGAAGCTCTGCGTTTCTGATCTGAAAACTGTAACCCATAGAAAAGCTCCCCAGGAGGTGCCTCACTCTGAGAAGAGATTTACAAGGAAGAGTGTGGTGGCTTCTCAGAGTTTCCAAGCAGGGAAACATTACTGGGAGGTGGACGGAGGACACAATAAAAGCGTGGCGCGTGTGGAGTGTGCCGGGATGATGTGGACAGGAGGAAGGAGTACGTGACTTTGTCTCCCGATCATGGGTACTGGGTCCTCAGACTGAATGGAGAACATTTGTATTTCACATTAAATCCCCGTTTTATCAGCGTCTTCCCCAGGACCCCACCTACAAAAATAGGGGTCTTCCTGGACTATGAGTGTGGGACCATCTCCTTCTTCAACATAAATGACCAGTCCCTTATTTATACCCTGACATGTCGGTTTGAAGGCTTATTGAGGCCCTACATTGAGTATCCGTCCTATAATGAGCAAAATGGAACTCCCAGAGACAAGCAACAGTGAGTCCTCCTCACAGGCAACCACGCCCTTCCTCCCCAGGGGTGAAATGTAGGATGAATCACATCCCACATTCTTCTTTAGGGATATTAAGGTCTCTCTCCCAGATCCAAAGTCCCGCAGCAGCCGGCCAAGGTGGCTTCCAGATGAAGGGGGACTGGCCTGTCCACATGGGAGTCAGGTGTCATGGCTGCCCTGAGCTGGGAGGGAAGAAGGCTGACATTACATTTAGTTTGCTCTCACTCCATCTGGCTAAGTGATCTTGAAATACCACCTCTCAGGTGAAGAACCGTCAGGAATTCCCATCTCACAGGCTGTGGTGTAGATTAAGTAGACAAGGAATGTGAATAATGCTTAGATCTTATTGATGACAGAGTGTATCCTAATGGTTTGTTCATTATATTACACTTTCAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:117), and/orATGGCTCTAATGCTCAGTTTGGTTCTGAGTCTCCTCAAGCTGGGATCAGGGCAGTGGCAGGTGTTTGGGCCAGACAAGCCTGTCCAGGCCTTGGTGGGGGAGGACGCAGCATTCTCCTGTTTCCTGTCTCCTAAGACCAATGCAGAGGCCATGGAAGTGCGGTTCTTCAGGGGCCAGTTCTCTAGCGTGGTCCACCTCTACAGGGACGGGAAGGACCAGCCATTTATGCAGATGCCACAGTATCAAGGCAGGACAAAACTGGTGAAGGATTCTATTGCGGAGGGGCGCATCTCTCTGAGGCTGGAAAACATTACTGTGTTGGATGCTGGCCTCTATGGGTGCAGGATTAGTTCCCAGTCTTACTACCAGAAGGCCATCTGGGAGCTACAGGTGTCAGCACTGGGCTCAGTTCCTCTCATTTCCATCACGGGATATGTTGATAGAGACATCCAGCTACTCTGTCAGTCCTCGGGCTGGTTCCCCCGGCCCACAGCGAAGTGGAAAGGTCCACAAGGACAGGATTTGTCCACAGACTCCAGGACAAACAGAGACATGCATGGCCTGTTTGATGTGGAGATCTCTCTGACCGTCCAAGAGAACGCCGGGAGCATATCCTGTTCCATGCGGCATGCTCATCTGAGCCGAGAGGTGGAATCCAGGGTACAGATAGGAGATACCTTTTTCGAGCCTATATCGTGGCACCTGGCTACCAAAGTACTGGGAATACTCTGCTGTGGCCTATTTTTTGGCATTGTTGGACTGAAGATTTTCTTCTCCAAATTCCAGTGGAAAATCCAGGCGGAACTGGACTGGAGAAGAAAGCACGGACAGGCAGAATTGAGAGACGCCCGGAAACACGCAGTGGAGGTGACTCTGGATCCAGAGACGGCTCACCCGAAGCTCTGCGTTTCTGATCTGAAAACTGTAACCCATAGAAAAGCTCCCCAGGAGGTGCCTCACTCTGAGAAGAGATTTACAAGGAAGAGTGTGGTGGCTTCTCAGAGTTTCCAAGCAGGGAAACATTACTGGGAGGTGGACGGAGGACACAATAAAAGGTGGCGCGTGGGAGTGTGCCGGGATGATGTGGACAGGAGGAAGGAGTACGTGACTTTGTCTCCCGATCATGGGTACTGGGTCCTCAGACTGAATGGAGAACATTTGTATTTCACATTAAATCCCCGTTTTATCAGCGTCTTCCCCAGGACCCCACCTACAAAAATAGGGGTCTTCCTGGACTATGAGTGTGGGACCATCTCCT-CTTCAACATAAATGACCAGTCCCTTATTTATACCCTGACATGTCGGTTTGAAGGCTTATTGAGGCCCTACATTGAGTATCCGTCCTATAATGAGCAAAATGGAACTCCCAGAGACAAGCAACAGTGA (SEQ ID NO:118). Polypeptides encoded bythese polynucleotides are also encompassed by the invention.

Preferred polypeptides of the invention comprise, or alternativelyconsist of the following amino acid sequence:

MALMLSLVLSLLKLGSGQWQVFGPDKPVQALVGEDAAFSCFLSPKTNAEAMEVRFFRGQFSSVVHLYRDGKDQPFMQMPQYQGRTKLVKDSIAEGRISLRLENITVLDAGLYGCRISSQSYYQKAIWELQVSALGSVPLISITGYVDRDIQLLCQSSGWFPRPTAKWKGPQGQDLSTDSRTNRDMHGLFDVEISLTVQENAGSISCSMRHAHLSREVESRVQIGDTFFEPISWHLATKVLGILCCGLFFGIVGLKIFFSKFQWKIQAELDWRRKHGQAELRDARKHAVEVTLDPETAHPKLCVSDLKTVTHRKAPQEVPHSEKRFTRKSVVASQSFQAGKHYWEVDGGHNKRWRVGVCRDDVDRRKEYVTLSPDHGYWVLRLNGEHLYFTLNPRFISVFPRTPPTKIGVFLDYECGTISFFNINDQSLIYTLTCRFEGLLRPYIEYPSYNEQNGTPRDKQQ(SEQ ID NO:119). Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

A preferred polynucleotide splice variant of the invention comprises thefollowing nucleic acid sequence:

ACCTTTTTCGAGCCTATATCGTGGCACCTGGCTACCAAAGTACTGGGAATACTCTGCTGTGGCCTATTTTTTGGCATTGTFGGACTGAAGATTTTCTTCTCCAAATTCCAGTGGAAAATCCAGGCGGAACTGGACTGGAGAAGAAAGCACGGACAGGCAGAATTGAGAGACGCCCGGAAACACGCAGTGGAGGTGACTCTGGATCCAGAGACGGCTCACCCGAAGCTCTGCGTTTCTGATCTGAAAACTGTAACCCATAGAAAAGCTCCCCAGGAGGTGCCTCACTCTGAGAAGAGATTTACAAGGAAGAGTGTGGTGGCTTCTCAGAGTTTCCAAGCAGGGAAACATTACTGGGAGGTGGACGGAGGACACAATAAAAGGTGGCGCGTGGGAGTGTGCCGGGATGATGTGGACAGGAGGAAGGAGTACGTGACTYTGTCTCCCGATCATGGGTACTGGGTCCTCAGACTGAATGGAGAACATTFGTATTTCACATTAAATCCCCGTTTTATCAGCGTCTTCCCCAGGACCCCACCTACAAAAATAGGGGTCTTCCTGGACTATGAGTGTGGGACCATCTCCTTCTTCAACATAAATGACCAGTCCCTTATTTATACCCTGACATGTCGGTTTGAAGGCTTATTGAGGCCCTACATTGAGTATCCGTCCTATAATGAGCAAAATGGAACTCCCAGAGACAAGCAACAGTGAGTCCTCCTCACAGGCAACCACGCCCTfCCTCCCCAGGGGTGAAATGTAGGATGAATCACATCCCACATTCTTCTTTAGGGATATTAAGGTCTCTCTCCCAGATCCAAAGTCCCGCAGCAGCCGGCCAAGGTGGCTTCCAGATGAAGGGGGACTGGCCTGTCCACATGGGAGTCAGGTGTCATGGCTGCCCTGAGCTGGGAGGGAAGAAGGCTGACATTACATTTAGTTTGCTCTCACTCCATCTGGCTAAGTGATCTTGAAATACCACCTCTCAGGTGAAGAACCGTCAGGAATTCCCATCTCACAGGCTGTGGTGTAGATTAAGTAGACAAGGAATGTGAATAATGCTTAGATCTTATTGATGACAGAGTGTATCCTAATGGTTTGTTCATTATATTACACTTTCAGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:120). Polypeptides encoded by thesepolynucleotides are also encompassed by the invention.

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, or all five ofthe immunogenic epitopes shown in SEQ ID NO: 37 as residues: Tyr-67 toPro-74, Ser-1 17 to Gln-123, Pro-161 to Met-185, Gly-224 to His-242,and/or Thr-299 to Trp-307. Polynucleotides encoding these polypeptidesare also encompassed by the invention, as are antibodies that bind oneor more of these polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

FIGS. 22A-C show the nucleotide (SEQ ID NO:19) and deduced amino acidsequence (SEQ ID NO:37) of BBIR II. Predicted amino acids from about 1to about 17 constitute the predicted signal peptide (amino acid residuesfrom about 1 to about 17 in SEQ ID NO:37) and are represented by theunderlined amino acid regions.

FIG. 23 shows the regions of similarity between the amino acid sequencesof the Butyrophlin and B7-like IgG superfamily receptor (BBIR II)protein (SEQ ID NO:37) and the bovine butyrophilin precursor (SEQ IDNO:121)

FIG. 24 shows an analysis of the integrin alpha 11 subunit (BBIR 11)amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicityand hydrophobicity; amphipathic regions; flexible regions; antigenicindex and surface probability are shown.

Translation products corresponding to this gene share homology to theB30.2-like domain which is characteristic of proteins containingzinc-binding B-box motifs, and particularly for butyrophilin familymembers. The polynucleotide contains an open reading frame encoding theBBIR II polypeptide of 318 amino acids. BBIR II exhibits a high degreeof homology at the amino acid level to the bovine butyrophilin precursor(as shown in FIG. 23). The present invention provides isolated nucleicacid molecules comprising a polynucleotide encoding the BBIR IIpolypeptide having the amino acid sequence shown in FIGS. 22A-C (SEQ IDNO:37). The nucleotide sequence shown in FIGS. 22A-C (SEQ ID NO:19) wasobtained by sequencing a cloned cDNA (HTTDB46), which was deposited onNovember 17 at the American Type Culture Collection, and given AccessionNumber 203484.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO: 19 is intended DNA fragments atleast about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:19. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:19. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Representative examples of BBIR II polynucleotide fragments ofthe invention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, from about 501 toabout 550, from about 551 to about 600, from about 601 to about 650,from about 651 to about 700, from about 701 to about 750, from about 751to about 800, from about 801 to about 850, from about 851 to about 900,from about 901 to about 950, from about 951 to about 1000, from about1001 to about 1050, from about 1051 to about 1100, from about 1101 toabout 1150, from about 1151 to about 1200, from about 1201 to about1250, from about 1251 to about 1300, from about 1301 to about 1350, fromabout 1351 to about 1400, from about 1401 to about 1450, from about 1451to about 1500, from about 1501 to about 1550, from about 1551 to about1600, from about 1601 to about 1650, from about 1651 to about 1700, fromabout 1701 to about 1750, from about 1751 to about 1800, from about 1801to about 1850, from about 1851 to about 1900, from about 1901 to about1950, from about 1951 to about 2000, from about 2001 to about 2050, fromabout 2051 to about 2100, from about 2101 to about 2150, from about 2151to about 2200, from about 2201 to about 2250, from about 2251 to about2300, from about 2301 to about 2350, from about 2351 to about 2400, fromabout 2401 to about 2450, from about 2451 to about 2500, from about 2501to about 2550, from about 2551 to about 2600, from about 2601 to about2650, from about 2651 to about 2700, from about 2701 to about 2750, fromabout 2751 to about 2800, from about 2801 to about 2850, from about 2851to about 2900, from about 2901 to about 2950, from about 2951 to about3000, from about 3001 to about 3050, from about 3051 to about 3059 ofSEQ ID NO:19, or the complementary strand thereto, or the cDNA containedin the deposited gene. In this context “about” includes the particularlyrecited ranges, larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus or at both termini.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding a member selected from the group: apolypeptide comprising or alternatively, consisting of, the mature BBIRII protein (amino acid residues from about 18 to about 318 in FIGS.22A-C (amino acids from about 18 to about 318 in SEQ ID NO:37). Sincethe location of this form of the protein has been predicted by computeranalysis, one of ordinary skill would appreciate that the amino acidresidues constituting these domains may vary slightly (e.g., by about 1to 15 amino acid residues) depending on the criteria used to define thislocation. In additional embodiments, the polynucleotides of theinvention encode functional attributes of BBIR II.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions of BBIR II. The data representing the structural orfunctional attributes of BBIR II set forth in FIG. 24 and/or Table VIII,as described above, was generated using the various modules andalgorithms of the DNA*STAR set on default parameters. In a preferredembodiment, the data presented in columns VIII, IX, XIII, and XIV ofTable VIII can be used to determine regions of BBIR II which exhibit ahigh degree of potential for antigenicity. Regions of high antigenicityare determined from the data presented in columns VIII, IX, XIII, and/orXIV by choosing values which represent regions of the polypeptide whichare likely to be exposed on the surface of the polypeptide in anenvironment in which antigen recognition may occur in the process ofinitiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 24, butmay, as shown in Table VIII, be represented or identified by usingtabular representations of the data presented in FIG. 24. The DNA*STARcomputer algorithm used to generate FIG. 24 (set on the original defaultparameters) was used to present the data in FIG. 24 in a tabular format(See Table VIII). The tabular format of the data in FIG. 24 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 24 and in Table VIIIinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS.22A-C. As set out in FIG. 24 and in Table VIII, such preferred regionsinclude Gamier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, etc.) may still beretained. For example, the ability of shortened BBIR II muteins toinduce and/or bind to antibodies which recognize the complete or matureforms of the polypeptides generally will be retained when less than themajority of the residues of the complete or mature polypeptide areremoved from the N terminus. Whether a particular polypeptide lackingN-terminal residues of a complete polypeptide retains such immunologicactivities can readily be determined by routine methods described hereinand otherwise known in the art. It is not unlikely that an BBIR IImutein with a large number of deleted N-terminal amino acid residues mayretain some biological or immunogenic activities. In fact, peptidescomposed of as few as six BBIR II amino acid residues may often evoke animmune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the BBIR IIamino acid sequence shown in FIGS. 22A-C, up to the cystein residue atposition number 313 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-318 of FIGS. 22A-C, where n1 is aninteger from 2 to 313 corresponding to the position of the amino acidresidue in FIGS. 22A-C (which is identical to the sequence shown as SEQID NO:37). In another embodiment, N-terminal deletions of the BBIR IIpolypeptide can be described by the general formula n2-318, where n2 isa number from 2 to 313, corresponding to the position of amino acididentified in FIGS. 22A-C. N-terminal deletions of the BBIR IIpolypeptide of the invention shown as SEQ ID NO:37 include polypeptidescomprising the amino acid sequence of residues: N-terminal deletions ofthe BBIR II polypeptide of the invention shown as SEQ ID NO:37 includepolypeptides comprising the amino acid sequence of residues: A-2 toT-318; L-3 to T-318; M4 to T-318; L-5 to T-318; S-6to T-318; L-7 toT-318; V-8 to T-318; L-9 to T-318; S-10 to T-318; L-11 to T-318; L-12 toT-318; K-13 to T-318; L-14 to T-318; G-15 to T-318; S-16 to T-318; G-17to T-318; Q-18 to T-318; W-19 to T-318; Q-20 to T-318; V-21 to T-318;F-22 to T-318; G-23 to T-318; P-24 to T-318; D-25 to T-318; K-26 toT-318; P-27 to T-318; V-28 to T-318; Q-29 to T-318; A-30 to T-318; L-31to T-318; V-32 to T-318; G-33 to T-318; E-34 to T-318; D-35 to T-318;A-36 to T-318; A-37 to T-318; F-38 to T-318; S-39 to T-319; C-40 toT-319; F-41 to T-318; L-42 to T-318; S-43 to T-318; P-44 to T-318; K-45to T-318; T-46 to T-318; N-47 to T-318; A-48 to T-318; E-49 to T-318;A-50 to T-318; M-51 to T-318; E-52 to T-318; V-53 to T-318; R-54 toT-318; F-55 to T-318; F-56 to T-318; R-57 to T-318; G-58 to T-318; Q-59to T-318; F-60 to T-318; S-61 to T-318; S-62 to T-318; V-63 to T-318;V-64 to T-318; H-65 to T-318; L-66 to T-318; Y-67 to T-318; R-68 toT-318; D-69 to T-318; G-70 to T-318; K-71 to T-318; D-72 to T-318; Q-73to T-318; P-74 to T-318; F-75 to T-318; M-76 to T-318; Q-77 to T-318;M-78 to T-318; P-79 to T-318; Q-80 to T-318; Y-81 to T-318; Q-82 toT-318; G-83 to T-318; R-84 to T-318; T-85 to T-318; K-86 to T-318; L-87to T-318; V-88 to T-318; K-89 to T-318; D-90 to T-318; S-91 to T-318;I-92 to T-318; A-93 to T-318; E-94 to T-318; G-95 to T-318; R-96 toT-318; I-97 to T-318; S-98 to T-318; L-99 to T-318; R-100 to T-318;L-101 to T-318; E-102 to T-318; N-103 to T-318; I-104 to T-318; T-105 toT-318; V-106 to T-318; L-107 to T-318; D-108 to T-318; A-109 to T-318;G-110 to T-318; L-111 to T-318; Y-112 to T-318; G-113 to T-318; C-114 toT-318; R-115 to T-318; I-116 to T-318; S-117 to T-318; S-118 to T-318;Q-119 to T-318; S-120to T-318; Y-121 to T-318; Y-122 to T-318; Q-123 toT-318; K-124 to T-318; A-125 to T-318; I-126 to T-318; W-127 to T-318;E-128 to T-318; L-129 to T-318; Q-130 to T-318; V-131 to T-318; S-132 toT-318; A-133 to T-318; L-134 to T-318; G-135 to T-318; S-136 to T-318;V-137 to T-318; P-138 to T-318; L-139 to T-318; I-140 to T-318; S-141 toT-318; I-142 to T-318; A-143 to T-318; G-144 to T-318; Y-145 to T-318;V-146 to T-318; D-147 to T-318; R-148 to T-318; D-149 to T-318; I-150 toT-318; Q-151 to T-318; L-152 to T-318; L-153 to T-318; C-154 to T-318;Q-155 to T-318; S-156 to T-318; S-157 to T-318; G-158 to T-318; W-159 toT-318; F-160 to T-318; P-161 to T-318; R-162 to T-318; P-163 to T-318;T-164 to T-318; A-165 to T-318; K-166 to T-318; W-167 to T-318; K-168 toT-318; G-169 to T-318; P-170 to T-318; Q-171 to T-318; G-172 to T-318;Q-173 to T-318; D-174 to T-318; L-175 to T-318; S-176 to T-318; T-177 toT-318; D-178 to T-318; S-179 to T-318; R-180 to T-318; T-181 to T-318;N-182 to T-318; R-183 to T-318; D-184 to T-318; M-185 to T-318; H-186 toT-318; G-187 to T-318; L-188 to T-318; F-189 to T-318; D-190 to T-318;V-191 to T-318; E-192 to T-318; I-193 to T-318; S-194 to T-318; L-195 toT-318; T-196 to T-318; V-197 to T-318; Q-198 to T-318; E-199 to T-318;N-200 to T-318; A-201 to T-318; G-202 to T-318; S-203 to T-318; I-204 toT-318; S-205 to T-318; C-206 to T-318; S-207 to T-318; M-208 to T-318;R-209 to T-318; H-210 to T-318; A-211 to T-318; H-212 to T-318; L-213 toT-318; S-214 to T-318; R-215 to T-318; E-216 to T-318; V-217 to T-318;E-218 to T-318; S-219 to T-318; R-220 to T-318; V-221 to T-318; Q-222 toT-318; I-223 to T-318; G-224 to T-318; D-225 to T-318; W-226 to T-318;R-227 to T-318; R-228 to T-318; K-229 to T-318; H-230 to T-318; G-231 toT-318; Q-232 to T-318; A-233 to T-318; G-234 to T-318; K-235 to T-318;R-236 to T-318; K-237 to T-318; Y-238 to T-318; S-239 to T-318; S-240 toT-318; S-241 to T-318; H-242 to T-318; I-243 to T-318; Y-244 to T-318;D-245 to T-318; S-246 to T-318; F-247 to T-318; P-248 to T-318; S-249 toT-318; L-250 to T-318; S-251 to T-318; F-252 to T-318; M-253 to T-318;D-254 to T-318; F-255 to T-318; Y-256 to T-318; I-257 to T-318; L-258 toT-318; R-259 to T-318; P-260 to T-318; V-261 to T-318; G-262 to T-318;P-263 to T-318; C-264 to T-318; R-265 to T-318; A-266 to T-318; K-267 toT-318; L-268 to T-318; V-269 to T-318; M-270 to T-318; G-271 to T-318;T-272 to T-318; L-273 to T-318; K-274 to T-318; L-275 to T-318; Q-276 toT-318; I-277 to T-318; L-278 to T-318; G-279 to T-318; E-280 to T-318;V-281 to T-318; H-282 to T-318; F-283 to T-318; V-284 to T-318; E-285 toT-318; K-286 to T-318; P-287 to T-318; H-288 to T-318; S-289 to T-318;L-290 to T-318; L-291 to T-318; Q-292 to T-318; I-293 to T-318; S-294 toT-318; G-295 to T-318; G-296 to T-318; S-297 to T-318; T-298 to T-318;T-299 to T-318; L-300 to T-318; K-301 to T-318; K-302 to T-318; G-303 toT-318; P-304 to T-318; N-305 to T-318; P-306 to T-318; W-307 to T-318;S-308 to T-318; F-309 to T-318; P-310 to T-318; S-311 to T-318; P-312 toT-318; C-313 to T-318; of SEQ ID NO:37. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g., fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities (e.g., ability to illicit mitogenicactivity, induce differentiation of normal or malignant cells, abilityto multimerize, etc.) may still be retained. For example the ability ofthe shortened BBIR II mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an BBIR II mutein with a large numberof deleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as six BBIRII amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the BBIR II polypeptide shown in FIGS. 22A-C, up to theserine residue at position number 6,, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m1 of FIG. 1, where m1is an integer from 6 to 318 corresponding to the position of the aminoacid residue in FIGS. 22A-C. Moreover, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of C-terminal deletions of theBBIR II polypeptide of the invention shown as SEQ ID NO:37 includepolypeptides comprising the amino acid sequence of residues M-1 toP-317; M-1 to F-316; M-1 to L-315; M-1 to A-314; M-1 to C-313; M-1 toP-312; M-1 to S-311; M-1 to P-310; M-1 to F-309; M-1 to S-308; M-1 toW-307; M-1 to P-306; M-1 to N-305; M-1 to P-304; M-1 to G-303; M-1 toK-302; M-1 to K-301; M-1 to L-300; M-1 to T-299; M-1 to T-298; M-1 toS-297; M-1 to G-296; M-1 to G-295; M-1 to S-294; M-1 to I-293; M-1 toQ-292; M-1 to L-291; M-1 to L-290; M-1 to S-289; M-1 to H-288; M-1 toP-287; M-1 to K-286; M-1 to E-285; M-1 to V-284; M-1 to F-283; M-1 toH-282; M-1 to V-281; M-1 to E-280; M-1 to G-279; M-1 to L-278; M-1 toI-277; M-1 to Q-276; M-1 to L-275; M-1 to K-274; M-1 to L-273; M-1 toT-272; M-1 to G-271; M-1 to M-270; M-1 to V-269; M-1 to L-268; M-1 toK-267; M-1 to A-266; M-1 to R-265; M-1 to C-264; M-1 to P-263; M-1 toG-262; M-1 to V-261; M-1 to P-260; M-1 to R-259; M-1 to L-258; M-1 toI-257; M-1 to Y-256; M-1 to F-255; M-1 to D-254; M-1 to M-253; M-1 toF-252; M-1 to S-251; M-1 to L-250; M-1 to S-249; M-1 to P-248; M-1 toF-247; M-1 to S-246; M-1 to D-245; M-1 to Y-244; M-1 to I-243; M-1 toH-242; M-1 to S-241; M-1 to S-240; M-1 to S-239; M-1 to Y-238; M-1 toK-237; M-1 to R-236; M-1 to K-235; M-1 to G-234; M-1 to A-233; M-1 toQ-232; M-1 to G-231; M-1 to H-230; M-1 to K-229; M-1 to R-228; M-1 toR-227; M-1 to W-226; M-1 to D-225; M-1 to G-224; M-1 to I-223; M-1 toQ-222; M-1 to V-221; M-1 to R-220; M-1 to S-219; M-1 to E-218; M-1 toV-217; M-1 to E-216; M-1 to R-215; M-1 to S-214; M-1 to L-213; M-1 toH-212; M-1 to A-211; M-1 to H-210; M-1 to R-209; M-1 to M-208; M-1 toS-207; M-1 to C-206; M-1 to S-205; M-1 to I-204; M-1 to S-203; M-1 toG-202; M-1 to A-201; M-1 to N-200; M-1 to E-199; M-1 to Q-198; M-1 toV-197; M-1 to T-196; M-1 to L-195; M-1 to S-194; M-1 to I-193; M-1 toE-192; M-1 to V-191; M-1 to D-190; M-1 to F-189; M-1 to L-188; M-1 toG-187; M-1 to H-186; M-1 to M-185; M-1 to D-184; M-1 to R-183; M-1 toN-182; M-1 to T-181; M-1 to R-180; M-1 to S-179; M-1 to D-178; M-1 toT-177; M-1 to S-176; M-1 to L-175; M-1 to D-174; M-1 to Q-173; M-1 toG-172; M-1 to Q-171; M-1 to P-170; M-1 to G-169; M-1 to K-168; M-1 toW-167; M-1 to K-166; M-1 to A-165; M-1 to T-164; M-1 to P-163; M-1 toR-162; M-1 to P-161; M-1 to F-160; M-1 to W-159; M-1 to G-158; M-1 toS-157; M-1 to S-156; M-1 to Q-155; M-1 C-154; M-1 to L-153; M-1 toL-152; M-1 to Q-151; M-1 to I-150; M-1 to D-149; M-1 to R-148; M-1 toD-147; M-1 to V-146; M-1 to Y-145; M-1 to G-144; M-1 to A-143; M-1 toI-142; M-1 to S-141; M-1 to I-140; M-1 to L-139; M-1 to P-138; M-1 toV-137; M-1 to S-136; M-1 to G-135; M-1 to L-134; M-1 to A-133; M-1 toS-132; M-1 to V-131; M-1 to Q-130; M-1 to L-129; M-1 to E-128; M-1 toW-127; M-1 to I-126; M-1 to A-125; M-1 to K-124; M-1 to Q-123; M-1 toY-122; M-1 to Y-121; M-1 to S-120; M-1 to Q-119; M-1 to S-118; M-1 toS-117; M-1 to I-116; M-1 to R-115; M-1 to C-114; M-1 to G-113; M-1 toY-112; M-1 to L-111; M-1 to G-110; M-1 to A-109; M-1 to D-108; M-1 toL-107; M-1 to V-106; M-1 to T-105; M-1 to I-104; M-1 to N-103; M-1 toE-102; M-1 to L-101; M-1 to R-100; M-1 to L-99; M-1 to S-98; M-1 toI-97; M-1 to R-96; M-1 to G-95; M-1 to E-94; M-1 to A-93; M-1 to I-92;M-1 to S-91; M-1 to D-90; M-1 to K-89; M-1 to V-88; M-1 to L-87; M-1 toK-86; M-1 to T-85; M-1 to R-84; M-1 to G-83; M-1 to Q-82; M-1 to Y-81;M-1 to Q-80; M-1 to P-79; M-1 to M-78; M-1 to Q-77; M-1 to M-76; M-1 toF-75; M-1 to P-74; M-1 to Q-73; M-1 to D-72; M-1 to K-71; M-1 to G-70;M-1 to D-69; M-1 to R-68; M-1 to Y-67; M-1 to L-66; M-1 to H-65; M-1 toV-64; M-1 to V-63; M-1 to S-62; M-1 to S-61; M-1 to F-60; M-1 to Q-59;M-1 to G-58; M-1 to R-57; M-1 to F-56; M-1 to F-55; M-1 to R-54; M-1 toV-53; M-1 to E-52; M-1 to M-51; M-1 to A-50; M-1 to E-49; M-1 to A-48;M-1 to N-47; M-1 to T-46; M-1 to K-45; M-1 to P-44; M-1 to S-43; M-1 toL-42; M-1 to F-41; M-1 to C-40; M-1 to S-39; M-1 to F-38; M-1 to A-37;M-1 to A-36; M-1 to D-35; M-1 to E-34; M-1 to G-33; M-1 to V-32; M-1 toL-31; M-1 to A-30; M-1 to Q-29; M-1 to V-28; M-1 to P-27; M-1 to K-26;M-1 to D-25; M-1 to P-24; M-1 to G-23; M-1 to F-22; M-1 to V-21; M-1 toQ-20; M-1 to W-19; M-1 to Q-18; M-1 to G-17; M-1 to S-16; M-1 to G-15;M-1 to L-14; M-1 to K-13; M-1 to L-11; M-1 to S-10; M-1 to L-9; M-1 toV-8; M-1 to L-7; M-1 to S-6; of SEQ ID NO:37. Polynucleotides encodingthese polypeptides are also encompassed by the invention, as areantibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g., fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:19 whichhave been determined from the following related cDNA genes: HTTDB46R(SEQ ID NO:122), and HSIEA44R (SEQ ID NO:123).

A polynucleotide encoding a polypeptide of the present invention isobtained from human small intestine, colon tumor, and human testes tumorcells and tissues. The polynucleotide of this invention was discoveredin a human testes tumor cDNA library.

Based on the sequence similarity to the bovin butyrophilin precursor,translation product of this gene is expected to share at least somebiological activities with B30.2-like domain containing proteins, andspecifically butyrophilin proteins. Such activities are known in theart, some of which are described elsewhere herein. Specifically,polynucleotides and polypeptides of the invention are also useful formodulating the differentiation of normal and malignant cells, modulatingthe proliferation and/or differentiation of cancer and neoplastic cells,and regulation of cell growth and differentiation. Polynucleotides andpolypeptides of the invention may represent a diagnostic marker forbreast diseases and/or disorders, in addition to disorders of secretoryorgans and tissues (which include, testicular and gastrointestinaldisorders, particularly those cells which serve secretory functions forseminal fluid or gastrointestinal hormones, and disorders of the mucosalmembranes of such cells and tissues, etc.).

The full-length protein should be a secreted protein, based uponhomology to the butyrophilin family of proteins. Therefore, it issecreted into milk, serum, urine, seminal fluid, or feces and thus thelevels is assayable from patient samples. Assuming specific expressionlevels are reflective of the presence of breast disorders (i.e., breastcancer, breast dysfunction, etc.) this protein would provide aconvenient diagnostic for early detection of such disorders

In addition, expression of this gene product may also be linked to theprogression of breast diseases, and therefore may itself actuallyrepresent a therapeutic or therapeutic target for the treatment ofbreast cancer. Polynucleotides and polypeptides of the invention mayplay an important role in the pathogenesis of human cancers and cellulartransformation, particularly those of secretory cells and tissues.Polynucleotides and polypeptides of the invention may also be involvedin the pathogenesis of developmental abnormalities based upon itspotential effects on proliferation and differentiation of cells andtissue cell types.

Due to the potential proliferating and differentiating activity of saidpolynucleotides and polypeptides, the invention is useful as atherapeutic agent in inducing tissue regeneration, for treatinginflammatory conditions. Moreover, the invention is useful in modulatingthe immune response to aberrant polypeptides, as may exist in rapidlyproliferating cells and tissue cell types, particularly in cancers. Theinvention, including agonists and/or antagonists thereof, is useful inmodulating the nutritional value of milk, its caloric content, its fatcontent, and may conceivably be useful in mediating the adaption ofbreast secretory function as a delivery vehicle for therapeutics (i.e.,transgenic breast secretory tissue for transferring therapeuticallyactive proteins to infants).

Alternatively, the expression within cellular sources marked byproliferating cells indicates this protein may play a role in theregulation of cellular division, and may show utility in the diagnosis,treatment, and/or prevention of developmental diseases and disorders,including cancer, and other proliferative conditions. Representativeuses are described in the “Hyperproliferative Disorders” and“Regeneration” sections below and elsewhere herein. Briefly,developmental tissues rely on decisions involving cell differentiationand/or apoptosis in pattern formation.

Dysregulation of apoptosis can result in inappropriate suppression ofcell death, as occurs in the development of some cancers, or in failureto control the extent of cell death, as is believed to occur in acquiredimmunodeficiency and certain neurodegenerative disorders, such as spinalmuscular atrophy (SMA).

Alternatively, this gene product is involved in the pattern of cellularproliferation that accompanies early embryogenesis. Thus, aberrantexpression of this gene product in tissues—particularly adulttissues—may correlate with patterns of abnormal cellular proliferation,such as found in various cancers. Because of potential roles inproliferation and differentiation, this gene product may haveapplications in the adult for tissue regeneration and the treatment ofcancers. It may also act as a morphogen to control cell and tissue typespecification. Therefore, the polynucleotides and polypeptides of thepresent invention are useful in treating, detecting, and/or preventingsaid disorders and conditions, in addition to other types ofdegenerative conditions. Thus this protein may modulate apoptosis ortissue differentiation and is useful in the detection, treatment, and/orprevention of degenerative or proliferative conditions and diseases. Theprotein is useful in modulating the immune response to aberrantpolypeptides, as may exist in proliferating and cancerous cells andtissues. The protein can also be used to gain new insight into theregulation of cellular growth and proliferation. Furthermore, theprotein may also be used to determine biological activity, to raiseantibodies, as tissue markers, to isolate cognate ligands or receptors,to identify agents that modulate their interactions, in addition to itsuse as a nutritional supplement. Protein, as well as, antibodiesdirected against the protein may show utility as a tumor marker and/orimmunotherapy targets for the above listed tissues.

This gene is expressed primarily in small intestine, colon tumor, and toa lesser extent in human testes tumor cells.

Therefore, polynucleotides and polypeptides of the invention are usefulas reagents for differential identification of the tissue(s) or celltype(s) present in a biological sample and for diagnosis of diseases andconditions which include, but are not limited to, gastrointestinaldiseases and/or disorders, in addition to lactation disorders, andtumors of the testes. Similarly, polypeptides and antibodies directed tothese polypeptides are useful in providing immunological probes fordifferential identification of the tissue(s) or cell type(s). For anumber of disorders of the above tissues or cells, particularly of theimmune and reproductive systems, expression of this gene atsignificantly higher or lower levels is routinely detected in certaintissues or cell types (e.g. immune, testicular, gastrointestinal, andcancerous and wounded tissues) or bodily fluids (e.g. lymph, serum,plasma, urine, synovial fluid and spinal fluid) or another tissue orcell sample taken from an individual having such a disorder, relative tothe standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

Features of Protein Encoded by Gene No: 10

The translation product of this gene contains a serine protease motifand accordingly is believed to possess serine protease activity. Assaysfor determining such activity are well known in the art. Preferredpolypeptides of this invention possess such activity.

Included in this invention as preferred domains are serine proteasehistidine active site domains, which were identified using the ProSiteanalysis tool (Swiss Institute of Bioinformatics). The catalyticactivity of the serine proteases from the trypsin family is provided bya charge relay system involving an aspartic acid residue hydrogen-bondedto a histidine, which itself is hydrogen-bonded to a serine. Thesequences in the vicinity of the active site serine and histidineresidues are well conserved in this family of proteases [1]. Consensuspattern: [LIVM]-[ST]-A-[STAG]-H-C, H is the active site residue.

Preferred polypeptides of the invention comprise, or alternativelyconsist of, the following amino acid sequence:GTLVAEKHVLTAAHCIHDGKTYVKGTQ (SEQ ID NO: 124). Polynucleotides encodingthese polypeptides are also encompassed by the invention, as areantibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g., fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

Further preferred are polypeptides comprising the serine proteasehistidine active site domain of the sequence referenced in Table XIV forthis gene, and at least 5, 10, 15, 20, 25, 30, 50, or 75 additionalcontiguous amino acid residues of this referenced sequence. Theadditional contiguous amino acid residues is N-terminal or C-terminal tothe serine protease histidine active site domain.

Alternatively, the additional contiguous amino acid residues is bothN-terminal and C-terminal to the serine protease histidine active sitedomain, wherein the total N- and C-terminal contiguous amino acidresidues equal the specified number. The above preferred polypeptidedomain is characteristic of a signature specific to serine proteaseproteins. Based on the sequence similarity, the translation product ofthis gene is expected to share at least some biological activities withserine proteases. Such activities are known in the art, some of whichare described elsewhere herein.

In another embodiment, polypeptides comprising the amino acid sequenceof the open reading frame upstream of the predicted signal peptide arecontemplated by the present invention. Specifically, polypeptides of theinvention comprise, or alternatively consist of, the following aminoacid sequence: GTRGQAWEPRALSRRPHLSERRSEPRPGRAARRGTVLGMAGIPGLLFLLFF

LLCAVGQVSPYSAPWKPTWPAYRLPVVLPQSTLNLAKPDFGAEAKLEVSSSCGPQCHKGTPLPTYEEAKQYLSYETLYANGSRTETQVGIYILSSSGDGAQHRDSGSSGKSRRKRQIYGYDSRFSIFGKDFLLNYPFSTSVKLSTGCTGTLVAEKHVLTAAHCIHDGKTYVKGTQKLRVGFLKPKFKDGGRGANDSTSAMPEQMKFQWIRVKRTHVPKGWIKGNANDIGMDYDYALLELKKPHKRKFMKIGVSPPAKQLPGGRIHFSGYDNDRPGNLVYRFCDVKDETYDLLYQQCDSQPGASGSGVYVRMWKRQHQKWERKIIGMISGHQWVDMDGSPQEFTRGCSEITPLQYIPDISIGV (SEQ ID NO:125). Polynucleotides encoding these polypeptides are also encompassedby the invention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

A preferred polypeptide variant of the invention comprises, oralternatively consists of, the following amino acid sequence:MAGIPGLLFLLFFLLCAVGQVSPYSAPWKPTWPAYRLPVVLPQSTLNLAKPD

FGAEAKLEVSSSCGPQCHKGTPLPTYEEAKQYLSYETLYANGSRTETQVGIYILSSSGDGAQHRDSGSSGKSRRKRQIYGYDSRFSIFGKDFLLNYPFSTSVKLSTGCTGTLVAEKHVLTAAHCIHDGKTYVKGTQKLRVGFLKPKFKDGGRGANDSTSAMPEQMKFQWIRVKRTHVPKGWIKGNANDIGMDYDYALLELKKPHKRKFMKIGVSPPAKQLPGGRIHFSGYDNDRPGNLVYRFCDVKDETYDLLYQQCDAQPGASGSGVYVRMWKRQQQKWERKIIGIFSGHQWVDMNGSPQDFNVAVRITPLKYAQICYWIKGNYLDCRE G (SEQ ID NO:126). Polynucleotides encoding these polypeptides are also encompassedby the invention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, or all thirteen of theimmunogenic epitopes shown in SEQ ID NO: 38 as residues: Pro-67 toThr-73, Pro-76 to Gln-83, Asn-93 to Thr-99, His-115 to Arg-128, His-178to Lys-189, Pro-197 to Ala-212, Val-224 to Trp-233, Lys-253 to Lys-259,Ser-280 to Asn-289, Asp-296 to Tyr-302, Gln-308 to Ala-315, Arg-327 toLys-335, and/or Asp-349 to Gly-358. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g., fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

FIGS. 25 A-B show the nucleotide (SEQ ID NO:20) and deduced amino acidsequence (SEQ ID NO:38) of the present invention. Predicted amino acidsfrom about 1 to about 19 constitute the predicted signal peptide (aminoacid residues from about 1 to about 19 in SEQ ID NO:38) and arerepresented by the underlined amino acid regions; amino acids from about162 to about 188 constitutes the predicted serine protease histidineactive site domain (amino acids residues from about 162 to about 188 inSEQ ID NO:38) and are represented by the double underlined amino acidregions; and amino acid residue 175 (amino acid residue 175 in SEQ IDNO:38) constitutes the predicted histidine active site residue and isrepresented by the bold amino acid.

FIG. 26 shows the regions of similarity between the amino acid sequencesof the present invention SEQ ID NO:38, and the Human Pancreatic Elastase2 protein (gi|219620)(SEQ ID NO: 127).

FIG. 27 shows an analysis of the amino acid sequence of SEQ ID NO:38.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the polypeptide having the aminoacid sequence shown in FIGS. 25A-B (SEQ ID NO:38), which was determinedby sequencing a cloned cDNA. The nucleotide sequence shown in FIGS.25A-B (SEQ ID NO:20) was obtained by sequencing a cloned cDNA (HUSAQ05),which was deposited on Nov. 17, 1998 at the American Type CultureCollection, and given Accession Number 203484. The deposited gene isinserted in the pSport plasmid (Life Technologies, Rockville, Md.) usingthe SalI/NotI restriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO:20 is intended DNA fragments atleast about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:20. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:20. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Representative examples of polynucleotide fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, and from about 501to about 550, and from about 551 to about 600, from about 601 to about650, from about 651 to about 700, from about 701 to about 750, fromabout 751 to about 800, from about 801 to about 850, from about 851 toabout 900, from about 901 to about 950, from about 951 to about 1000,from about 1001 to about 1050, from about 1051 to about 1100, from about1101 to about 1150, from about 1151 to about 1200, from about 1201 toabout 1250, from about 1251 to about 1300, from about 1301 to about1350, from about 1351 to about 1400, from about 1401 to about 1450, fromabout 1451 to about 1500, from about 1501 to about 1550, from about 1551to about 1600, from about 1601 to about 1650, from about 1651 to about1699 of SEQ ID NO:20, or the complementary strand thereto, or the cDNAcontained in the deposited gene. In this context “about” includes theparticularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini. In additionalembodiments, the polynucleotides of the invention encode functionalattributes of the corresponding protein.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions. The data representing the structural or functionalattributes of the protein set forth in FIG. 27 and/or Table IX, asdescribed above, was generated using the various modules and algorithmsof the DNA*STAR set on default parameters. In a preferred embodiment,the data presented in columns VIII, IX, XIII, and XIV of Table IX can beused to determine regions of the protein which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX, XIII, and/or XIV bychoosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

Certain preferred regions in these regards are set out in FIG. 27, butmay, as shown in Table IX, be represented or identified by using tabularrepresentations of the data presented in FIG. 27. The DNA*STAR computeralgorithm used to generate FIG. 27 (set on the original defaultparameters) was used to present the data in FIG. 27 in a tabular format(See Table IX). The tabular format of the data in FIG. 27 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 27 and in Table IXinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIG. 1. Asset out in FIG. 27 and in Table IX, such preferred regions includeGamier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, etc.) may still beretained. For example, the ability of shortened muteins to induce and/orbind to antibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a mutein with a large numberof deleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixamino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence shown in FIGS. 25A-B, up to the aspartic acid residue atposition number 370 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-375 of FIGS. 25A-B, where n1 is aninteger from 2 to 370 corresponding to the position of the amino acidresidue in FIGS. 25A-B (which is identical to the sequence shown as SEQID NO:38). N-terminal deletions of the polypeptide of the inventionshown as SEQ ID NO:38 include polypeptides comprising the amino acidsequence of residues: A-2 to V-375; G-3 to V-375; 14 to V-375; P-5 toV-375; G-6 to V-375; L-7 to V-375; L-8 to V-375; F-9 to V-375; L-10 toV-375; L-11 to V-375; F-12 to V-375; F-13 to V-375; L-14 to V-375; L-15to V-375; C-16 to V-375; A-17 to V-375; V-18 to V-375; G-19 to V-375;Q-20 to V-375; V-21 to V-375; S-22 to V-375; P-23 to V-375; Y-24 toV-375; S-25 to V-375; A-26 to V-375; P-27 to V-375; W-28 to V-375; K-29to V-375; P-30 to V-375; T-31 to V-375; W-32 to V-375; P-33 to V-375;A-34 to V-375; Y-35 to V-375; R-36 to V-375; L-37 to V-375; P-38 toV-375; V-39 to V-375; V-40 to V-375; L-41 to V-375; P-42 to V-375; Q-43to V-375; S-44 to V-375; T-45 to V-375; L-46 to V-375; N-47 to V-375;L-48 to V-375; A-49 to V-375; K-50 to V-375; P-51 to V-375; D-52 toV-375; F-53 to V-375; G-54 to V-375; A-55 to V-375; E-56 to V-375; A-57to V-375; K-58 to V-375; L-59 to V-375; E-60 to V-375; V-61 to V-375;S-62 to V-375; S-63 to V-375; S-64 to V-375; C-65 to V-375; G-66 toV-375; P-67 to V-375; Q-68 to V-375; C-69 to V-375; H-70 to V-375; K-71to V-375; G-72 to V-375; T-73 to V-375; P-74 to V-375; L-75 to V-375;P-76 to V-375; T-77 to V-375; Y-78 to V-375; E-79 to V-375; E-80 toV-375; A-81 to V-375; K-82 to V-375; Q-83 to V-375; Y-84 to V-375; L-85to V-375; S-86 to V-375; Y-87 to V-375; E-88 to V-375; T-89 to V-375;L-90 to V-375; Y-91 to V-375; A-92 to V-375; N-93 to V-375; G-94 toV-375; S-95 to V-375; R-96 to V-375; T-97 to V-375; E-98 to V-375; T-99to V-375; Q-100 to V-375; V-101 to V-375; G-102 to V-375; I-103 toV-375; Y-104 to V-375; I-105 to V-375; L-106 to V-375; S-107 to V-375;S-108 to V-375; S-109 to V-375; G-110 to V-375; D-111 to V-375; G-112 toV-375; A-113 to V-375; Q-114 to V-375; H-115 to V-375; R-116 to V-375;D-117 to V-375; S-118 to V-375; G-119 to V-375; S-120 to V-375; S-121 toV-375; G-122 to V-375; K-123 to V-375; S-124 to V-375; R-125 to V-375;R-126 to V-375; K-127 to V-375; R-128 to V-375; Q-129 to V-375; I-130 toV-375; Y-131 to V-375; G-132 to V-375; Y-133 to V-375; D-134 to V-375;S-135 to V-375; R-136 to V-375; F-137 to V-375; S-138 to V-375; I-139 toV-375; F-140 to V-375; G-141 to V-375; K-142 to V-375; D-143 to V-375;F-144 to V-375; L-145 to V-375; L-146 to V-375; N-147 to V-375; Y-148 toV-375; P-149 to V-375; F-150 to V-375; S-151 to V-375; T-152 to V-375;S-153 to V-375; V-154 to V-375; K-155 to V-375; L-156 to V-375; S-157 toV-375; T-158 to V-375; G-159 to V-375; C-160 to V-375; T-161 to V-375;G-162 to V-375; T-163 to V-375; L-164 to V-375; V-165 to V-375; A-166 toV-375; E-167 to V-375; K-168 to V-375; H-169 to V-375; V-170 to V-375;L-171 to V-375; T-172 to V-375; A-173 to V-375; A-174 to V-375; H-175 toV-375; C-176 to V-375; I-177 to V-375; H-178 to V-375; D-179 to V-375;G-180 to V-375; K-181 to V-375; T-182 to V-375; Y-183 to V-375; V-184 toV-375; K-185 to V-375; G-186 to V-375; T-187 to V-375; Q-188 to V-375;K-189 to V-375; L-190 to V-375; R-191 to V-375; V-192 to V-375; G-193 toV-375; F-194 to V-375; L-195 to V-375; K-196 to V-375; P-197 to V-375;K-198 to V-375; F-199 to V-375; K-200 to V-375; D-201 to V-375; G-202 toV-375; G-203 to V-375; R-204 to V-375; G-205 to V-375; A-206 to V-375;N-207 to V-375; D-208 to V-375; S-209 to V-375; T-210 to V-375; S-211 toV-375; A-212 to V-375; M-213 to V-375; P-214 to V-375; E-215 to V-375;Q-216 to V-375; M-217 to V-375; K-218 to V-375; F-219 to V-375; Q-220 toV-375; W-221 to V-375; I-222 to V-375; R-223 to V-375; V-224 to V-375;K-225 to V-375; R-226 to V-375; T-227 to V-375; H-228 to V-375; V-229 toV-375; P-230 to V-375; K-231 to V-375; G-232 to V-375; W-233 to V-375;I-234 to V-375; K-235 to V-375; G-236 to V-375; N-237 to V-375; A-238 toV-375; N-239 to V-375; D-240 to V-375; I-241 to V-375; G-242 to V-375;M-243 to V-375; D-244 to V-375; Y-245 to V-375; D-246 to V-375; Y-247 toV-375; A-248 to V-375; L-249 to V-375; L-250 to V-375; E-251 to V-375;L-252 to V-375; K-253 to V-375; K-254 to V-375; P-255 to V-375; H-256 toV-375; K-257 to V-375; R-258 to V-375; K-259 to V-375; F-260 to V-375;M-261 to V-375; K-262 to V-375; I-263 to V-375; G-264 to V-375; V-265 toV-375; S-266 to V-375; P-267 to V-375; P-268 to V-375; A-269 to V-375;K-270 to V-375; Q-271 to V-375; L-272 to V-375; P-273 to V-375; G-274 toV-375; G-275 to V-375; R-276 to V-375; I-277 to V-375; H-278 to V-375;F-279 to V-375; S-280 to V-375; G-281 to V-375; Y-282 to V-375; D-283 toV-375; N-284 to V-375; D-285 to V-375; R-286 to V-375; P-287 to V-375;G-288 to V-375; N-289 to V-375; L-290 to V-375; V-291 to V-375; Y-292 toV-375; R-293 to V-375; F-294 to V-375; C-295 to V-375; D-296 to V-375;V-297 to V-375; K-298 to V-375; D-299 to V-375; E-300 to V-375; T-301 toV-375; Y-302 to V-375; D-303 to V-375; L-304 to V-375; L-305 to V-375;Y-306 to V-375; Q-307 to V-375; Q-308 to V-375; C-309 to V-375; D-310 toV-375; S-311 to V-375; Q-312 to V-375; P-313 to V-375; G-314 to V-375;A-315 to V-375; S-316 to V-375; G-317 to V-375; S-318 to V-375; G-319 toV-375; V-320 to V-375; Y-321 to V-375; V-322 to V-375; R-323 to V-375;M-324 to V-375; W-325 to V-375; K-326 to V-375; R-327 to V-375; Q-328 toV-375; H-329 to V-375; Q-330 to V-375; K-331 to V-375; W-332 to V-375;E-333 to V-375; R-334 to V-375; K-335 to V-375; I-336 to V-375; I-337 toV-375; G-338 to V-375; M-339 to V-375; I-340 to V-375; S-341 to V-375;G-342 to V-375; H-343 to V-375; Q-344 to V-375; W-345 to V-375; V-346 toV-375; D-347 to V-375; M-348 to V-375; D-349 to V-375; G-350 to V-375;S-351 to V-375; P-352 to V-375; Q-353 to V-375; E-354 to V-375; F-355 toV-375; T-356 to V-375; R-357 to V-375; G-358 to V-375; C-359 to V-375;S-360 to V-375; E-361 to V-375; I-362 to V-375; T-363 to V-375; P-364 toV-375; L-365 to V-375; Q-366 to V-365; Y-367 to V-375; I-368 to V-375;P-369 to V-375; D-370 to V-375; of SEQ ID NO:38. Polynucleotidesencoding these polypeptides are also encompassed by the invention, asare antibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g., fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities such as ability to modulate theextracellular matrix etc.) may still be retained. For example theability to induce and/or bind to antibodies which recognize the completeor mature forms of the polypeptide generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the C-terminus. Whether a particular polypeptidelacking C-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata mutein with a large number of deleted C-terminal amino acid residuesmay retain some biological or immunogenic activities. In fact, peptidescomposed of as few as six amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the polypeptide shown in FIGS. 25A-B, up to the glycineresidue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m1 of FIGS. 25A-B,where m1 is an integer from 6 to 375 corresponding to the position ofthe amino acid residue in FIGS. 25A-B. Moreover, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, the amino acid sequence of C-terminal deletions of thepolypeptide of the invention shown as SEQ ID NO:38 include polypeptidescomprising the amino acid sequence of residues: M-1 to G-374; M-1 toI-373; M-1 to S-372; M-1 to I-371; M-1 to D-370; M-1 to P-369; M-1 toI-368; M-1 to Y-367; M-1 to Q-366; M-1 to L-365; M-1 to P-364; M-1 toT-363; M-1 to I-362; M-1 to E-361; M-1 to S-360; M-1 to C-359; M-1 toG-358; M-1 to R-357; M-1 to T-356; M-1 to F-355; M-1 to E-354; M-1 toQ-353; M-1 to P-352; M-1 to S-351; M-1 to G-350; M-1 to D-349; M-1 toM-348; M-1 to D-347; M-1 to V-346; M-1 to W-345; M-1 to Q-344; M-1 toH-343; M-1 to G-342; M-1 to S-341; M-1 to I-340; M-1 to M-339; M-1 toG-338; M-1 to I-337; M-1 to I-336; M-1 to K-335; M-1 to R-334; M-1 toE-333; M-1 to W-332; M-1 to K-331; M-1 to Q-330; M-1 to H-329; M-1 toQ-328; M-1 to R-327; M-1 to K-326; M-1 to W-325; M-1 to M-324; M-1 toR-323; M-1 to V-322; M-1 to Y-321; M-1 to V-320; M-1 to G-319; M-1 toS-318; M-1 to G-317; M-1 to S-316; M-1 to A-315; M-1 to G-314; M-1 toP-313; M-1 to Q-312; M-1 to S-311; M-1 to D-310; M-1 to C-309; M-1 toQ-308; M-1 to Q-307; M-1 to Y-306; M-1 to L-305; M-1 to L-304; M-1 toD-303; M-1 to Y-302; M-1 to T-301; M-1 to E-300; M-1 to D-299; M-1 toK-298; M-1 to V-297; M-1 to D-296; M-1 to C-295; M-1 to F-294; M-1 toR-293; M-1 to Y-292; M-1 to V-291; M-1 to L-290; M-1 to N-289; M-1 toG-288; M-1 to P-287; M-1 to R-286; M-1 to D-285; M-1 to N-284; M-1 toD-283; M-1 to Y-282; M-1 to G-281; M-1 to S-280; M-1 to F-279; M-1 toH-278; M-1 to I-277; M-1 to R-276; M-1 to G-275; M-1 to G-274; M-1 toP-273; M-1 to L-272; M-1 to Q-271; M-1 to K-270; M-1 to A-269; M-1 toP-268; M-1 to P-267; M-1 to S-266; M-1 to V-265; M-1 to G-264; M-1 toI-263; M-1 to K-262; M-1 to M-261; M-1 to F-260; M-1 to K-259; M-1 toR-258; M-1 to K-257; M-1 to H-256; M-1 to P-255; M-1 to K-254; M-1 toK-253; M-1 to L-252; M-1 to E-251; M-1 to L-250; M-1 to L-249; M-1 toA-248; M-1 to Y-247; M-1 to D-246; M-1 to Y-245; M-1 to D-244; M-1 toM-243; M-1 to G-242; M-1 to I-241; M-1 to D-240; M-1 to N-239; M-1 toA-238; M-1 to N-237; M-1 to G-236; M-1 to K-235; M-1 to I-234; M-1 toW-233; M-1 to G-232; M-1 to K-231; M-1 to P-230; M-1 to V-229; M-1 toH-228; M-1 to T-227; M-1 to R-226; M-1 to K-225; M-1 to V-224; M-1 toR-223; M-1 to I-222; M-1 to W-221; M-1 to Q-220; M-1 to F-219; M-1 toK-218; M-1 to M-217; M-1 to Q-216; M-1 to E-215; M-1 to P-214; M-1 toM-213; M-1 to A-212; M-1 to S-211; M-1 to T-210; M-1 to S-209; M-1 toD-208; M-1 to N-207; M-1 to A-206; M-1 to G-205; M-1 to R-204; M-1 toG-203; M-1 to G-202; M-1 to D-201; M-1 to K-200; M-1 to F-199; M-1 toK-198; M-1 to P-197; M-1 to K-196; M-1 to L-195; M-1 to F-194; M-1 toG-193; M-1 to V-192; M-1 to R-191; M-1 to L-190; M-1 to K-189; M-1 toQ-188; M-1 to T-187; M-1 to G-186; M-1 to K-185; M-1 to V-184; M-1 toY-183; M-1 to T-182; M-1 to K-181; M-1 to G-180; M-1 to D-179; M-1 toH-178; M-1 to I-177; M-1 to C-176; M-1 to H-175; M-1 to A-174; M-1 toA-173; M-1 to T-172; M-1 to L-171; M-1 to V-170; M-1 to H-169; M-1 toK-168; M-1 to E-167; M-1 to A-166; M-1 to V-165; M-1 to L-164; M-1 toT-163; M-1 to G-162; M-1 to T-161; M-1 to C-160; M-1 to G-159; M-1 toT-158; M-1 to S-157; M-1 to L-156; M-1 to K-155; M-1 to V-154; M-1 toS-153; M-1 to T-152; M-1 to S-151; M-1 to F-150; M-1 to P-149; M-1 toY-148; M-1 to N-147; M-1 to L-146; M-1 to L-145; M-1 to F-144; M-1 toD-143; M-1 to K-142; M-1 to G-141; M-1 to F-140; M-1 to I-139; M-1 toS-138; M-1 to F-137; M-1 to R-136; M-1 to S-135; M-1 to D-134; M-1 toY-133; M-1 to G-132; M-1 to Y-131; M-1 to I-130; M-1 to Q-129; M-1 toR-128; M-1 to K-127; M-1 to R-126; M-1 to R-125; M-1 to S-124; M-1 toK-123; M-1 to G-122; M-1 to S-121; M-1 to S-120; M-1 to G-119; M-1 toS-118; M-1 to D-117; M-1 to R-116; M-1 to H-115; M-1 to Q-114; M-1 toA-113; M-1 to G-112; M-1 to D-111; M-1 to G-110; M-1 to S-109; M-1 toS-108; M-1 to S-107; M-1 to L-106; M-1 to I-105; M-1 to Y-104; M-1 toI-103; M-1 to G-102; M-1 to V-101; M-1 to Q-100; M-1 to T-99; M-1 toE-98; M-1 to T-97; M-1 to R-96; M-1 to S-95; M-1 to G-94; M-1 to N-93;M-1 to A-92; M-1 to Y-91; M-1 to L-90; M-1 to T-89; M-1 to E-88; M-1 toY-87; M-1 to S-86; M-1 to L-85; M-1 to Y-84; M-1 to Q-83; M-1 to K-82;M-1 to A-81; M-1 to E-80; M-1 to E-79; M-1 to Y-78; M-1 to T-77; M-1 toP-76; M-1 to L-75; M-1 to P-74; M-1 to T-73; M-1 to G-72; M-1 to K-71;M-1 to H-70; M-1 to C-69; M-1 to Q-68; M-1 to P-67; M-1 to G-66; M-1 toC-65; M-1 to S-64; M-1 to S-63 M-1 to S-62; M-1 to V-61; M-1 to E-60;M-1 to L-59; M-1 to K-58; M-1 to A-57; M-1 to E-56; M-1 to A-55; M-1 toG-54; M-1 to F-53; M-1 to D-52; M-1 to P-51; M-1 to K-50; M-1 to A-49;M-1 to L-48; M-1 to N-47; M-1 to L-46; M-1 to T-45; M-1 to S-44; M-1 toQ-43; M-1 to P-42; M-1 to L-41; M-1 to V-40; M-1 to V-39; M-1 to P-38;M-1 to L-37; M-1 R-36; M-1 to Y-35; M-1 to A-34; M-1 to P-33; M-1 toW-32; M-1 to T-31; M-1 to P-30; M-1 to K-29; M-1 TO W-28; M-1 to P-27;M-1 to A-26; M-1 to S-25; M-1 to Y-24; M-1 to P-23; M-1 to S-22; M-1 toV-21; M-1 to Q-20; M-1 to M-1 G-19; M-1 to V-18; M-1 to A-17; M-1 toC-16; M-1 to L-15; M-1 to L-14; M-1 to F-13; M-1 to F-12; M-1 to L-11;M-1 to L-10; M-1 to F-9; M-1 to L-8; M-1 to L-7; M-1 to G-6; of SEQ IDNO:38. Polynucleotides encoding these polypeptides are also encompassedby the invention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:20 whichhave been determined from the following related cDNA genes: HFKCF40F(SEQ ID NO:128), HSRDF26R (SEQ ID NO:129), HTEBE07R (SEQ ID NO:130),HFTBP-82R (SEQ ID NO:131), HAQBJ11R (SEQ ID NO:132), HAFBB11R (SEQ IDNO:133), HOEFO85R (SEQ ID NO:134), and HUVGY95R (SEQ ID NO:135).

The gene encoding the disclosed cDNA is believed to reside on chromosome12. Accordingly, polynucleotides related to this invention are useful asa marker in linkage analysis for chromosome 12.

This gene is expressed primarily in endothelial cells, fibroblasts,smooth muscle, and osteoblasts, and to a lesser extent in brain, heart,placental tissues, lung, and many other tissues. Moreover, thetranscript is present in HUVEC, HUVEC+LPS, smooth muscle, fibroblasts;present in heart, brain, placenta, lung, liver, muscle, kidney,pancreas, spleen, thymus, prostate, testes, ovary, small intestine,colon and weakly in PBLs.

Northern analysis indicates that this gene is expressed highest inHUVEC, HUVEC+LPS, smooth muscle, fibroblasts, present in heart, brain,placenta, lung, liver, muscle, kidney, pancreas, spleen, thymus,prostate, testes, ovary, small intestine, colon and weakly in PBLs.

Therefore, polynucleotides and polypeptides of the invention, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of diseases and conditions which include, but are not limitedto, disorders of vascularized tissues. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the vascular tissues, expression of this gene atsignificantly higher or lower levels is routinely detected in certaintissues or cell types (e.g. vascular, skeletal, developmental, neural,cardiovascular, pulmonary, renal, immune, hematopoietic, reproductive,gastrointestinal, and cancerous and wounded tissues) or bodily fluids(e.g., lymph, seminal, fluid, amniotic fluid, serum, plasma, urine,synovial fluid and spinal fluid) or another tissue or cell sample takenfrom an individual having such a disorder, relative to the standard geneexpression level, i.e., the expression level in healthy tissue or bodilyfluid from an individual not having the disorder.

The tissue distribution in the vascularized endothelial cells indicatesthat polynucleotides and polypeptides corresponding to this gene areuseful for the diagnosis and treatment of diseases of vascularizedtissues, such as atherosclerosis, ataxia malabsortion, andhyperlipidemia. These and other factors often result in othercardiovascular disease. Furthermore, translation product of this gene isuseful for the treatment of wounds, and may facilitate the wound healingprocess. Moreover, the protein is useful in the detection, treatment,and/or prevention of a variety of vascular disorders and conditions,which include, but are not limited to miscrovascular disease, vascularleak syndrome, aneurysm, stroke, embolism, thrombosis, coronary arterydisease, arteriosclerosis, and/or atherosclerosis. Based upon the tissuedistribution of this protein, antagonists directed against this proteinis useful in blocking the activity of this protein. Accordingly,preferred are antibodies which specifically bind a portion of thetranslation product of this gene.

Also provided is a kit for detecting tumors in which expression of thisprotein occurs. Such a kit comprises in one embodiment an antibodyspecific for the translation product of this gene bound to a solidsupport. Also provided is a method of detecting these tumors in anindividual which comprises a step of contacting an antibody specific forthe translation product of this gene to a bodily fluid from theindividual, preferably serum, and ascertaining whether antibody binds toan antigen found in the bodily fluid. Preferably the antibody is boundto a solid support and the bodily fluid is serum. The above embodiments,as well as other treatments and diagnostic tests (kits and methods), aremore particularly described elsewhere herein. Furthermore, the proteinmay also be used to determine biological activity, to raise antibodies,as tissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:20 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1685 of SEQID NO:20, b is an integer of 15 to 1699, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:20, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 11

Translation products corresponding to this gene share sequence homologywith Cytotoxic-Regulatory T-Cell Associated Molecule (CRTAM) protein,which is thought to be important in the regulation of celluarphysiology, development, differentiation or function of various celltypes, including haematopoietic cells and various T-cell progenitors.See for example, International Publication No. WO 96/34102 incorporatedherein by reference in its entirety. Moreover, translation productscorresponding to this gene also share sequence homology with thethymocyte activation and developmental protein and the class-IMHC-restricted T cell associated molecule (See Genbank Accession Nos.gi|2665790, gb|AAB88491.1, gb|AAC80267.1, and gi|3930163; allinformation and references contained within these accessions are herebyincorporated herein by reference). Based on the sequence similarity, thetranslation product of this gene is expected to share at least somebiological activities with T-cell modulatory proteins. Such activitiesare known in the art, some of which are described elsewhere herein.

Preferred polypeptides of the invention comprise, or alternativelyconsist of, the following amino acid sequence:MASVVLPSGSQCAAAAAAAAPPGLRLRLLLLLFSAAALIPTGDGQNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQPVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDYMLYVYDPPTTIPPPTTrTTTTTrTILTIITDSRAGEEGSRAVDHAVIGGVVAVVVFAMLCLLIILGRYFARHKGTYFTHEAKGADDAADADTAIINAEGGQNNSEEKKEYFI (SEQ ID NO: 136).Polynucleotides encoding these polypeptides are also encompassed by theinvention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

The polypeptide of this latter embodiment has been determined to have atransmembrane domain at about amino acid position 379-395 of the aminoacid sequence referenced in Table XIV for this gene. Moreover, acytoplasmic tail encompassing amino acids 396 to 442 of this protein hasalso been determined. Based upon these characteristics, it is believedthat the protein product of this gene shares structural features to typela membrane proteins.

Preferred polynucleotides comprise, or alternatively consist of, thefollowing nucleic acid sequence: ATGGCGAGTGTAGTGC

TGCCGAGCGGATCCCAGTGTGCGGCGGCAGCGGCGGCGGCGGCGCCTCCCGGGCTCCGGCTCCGGCTTCTGCTGTTGCTCTTCTCCGCCGCGGCACTGATCCCCACAGGTGATGGGCAGAATCTGTTTACGAAAGACGTGACAGTGATCGAGGGAGAGGTTGCGACCATCAGTTGCCAAGTCAATAAGAGTGACGACTCTGTGATTCAGCTACTGAATCCCAACAGGCAGACCATTTATTTCAGGGACTTCAGGCCTTTGAAGGACAGCAGGTTTCAGTTGCTGAATFTTTCTAGCAGTGAACTCAAAGTATCATTGACAAACGTCTCAATTTCTGATGAAGGAAGATACTTTTGCCAGCTCTATACCGATCCCCCACAGGAAAGTTACACCACCATCACAGTCCTGGTCCCACCACGTAATCTGATGATCGATATCCAGAAAGACACTGCGGTGGAAGGTGAGGAGATTGAAGTCAACTGCACTGCTATGGCCAGCAAGCCAGCCACGACTATCAGGTGGTTCAAAGGGAACACAGAGCTAAAAGGCAAATCGGAGGTGGAAGAGTGGTCAGACATGTACACTGTGACCAGTCAGCTGATGCTGAAGGTGCACAAGGAGGACGATGGGGTCCCAGTGATCTGCCAGGTGGAGCACCCTGCGGTCACTGGAAACCTGCAGACCCAGCGGTATCTAGAAGTACAGTATAAGCCTCAAGTGCACATTCAGATGACTTATCCTCTACAAGGCTTAACCCGGGAAGGGGACGCGCTTGAGTTAACATGTGAAGCCATCGGGAAGCCCCAGCCTGTGATGGTAACTTGGGTGAGAGTCGATGATGAAATGCCTCAACACGCCGTACTGTCTGGGCCCAACCTGTTCATCAATAACCTAAACAAAACAGATAATGGTACATACCGCTGTGAAGCTTCAAACATAGTGGGGAAAGCTCACTCGGATTATATGCTGTATGTATACGATCCCCCCACAACTATCCCTCCTCCCACAACAACCACCACCACCACCACCACCACCACCACCACCATCCTTACCATCATCACAGATTCCCGAGCAGGTGAAGAAGGCTCGATCAGGGCAGTGGATCATGCCGTGATCGGTGGCGTCGTGGCGGTGGTGGTGTTCGCCATGCTGTGCTTGCTCATCATTCTGGGGCGCTATmGCCAGACATAAAGGTACATACTTCACTCATGAAGCCAAAGGAGCCGATGACGCAGCAGACGCAGACACAGCTATAATCAATGCAGAAGGAGGACAGAACAACTCCGAAGAAAAGAAAGAGTACTTCATCTAG (SEQ ID NO:137). Alsopreferred are the polypeptides encoded by these polynucleotides.

Preferred polypeptides of the present invention comprise, oralternatively consist of, one, two, three, four, five, six, seven,eight, nine, or all nine of the immunogenic epitopes shown in SEQ ID NO:39 as residues: Gly-42 to Phe-48, Val-66 to Asp-71, Asn-78 to Thr-83,Asp-88 to Arg-96, Tyr-127 to Tyr-135, Lys-181 to Trp-195, His-210 toGly-215, Leu-303 to Thr-310, and/or Thr-341 to Thr-350. Polynucleotidesencoding these polypeptides are also encompassed by the invention, asare antibodies that bind one or more of these polypeptides. Moreover,fragments and variants of these polypeptides (e.g., fragments asdescribed herein, polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identical to these polypeptides and polypeptides encoded bythe polynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

FIGS. 28A-B shows the nucleotide (SEQ ID NO:21) and deduced amino acidsequence (SEQ ID NO:39) of the present invention. Predicted amino acidsfrom about 1 to about 44 constitute the predicted signal peptide (aminoacid residues from about 1 to about 44 in SEQ ID NO:39) and arerepresented by the underlined amino acid regions.

FIG. 29 shows the regions of similarity between the amino acid sequencesof the present invention SEQ ID NO:39, the human poliovirus receptorprotein (gi|1524088) (SEQ ID NO: 138), the human class-I MHC-restrictedT cell associated molecule (WO9634102) (SEQ ID NO:144), and the Gallusgallus thymocyte activation and developmental protein(gb|AAB88491.1)(SEQ ID NO:145).

FIG. 30 shows an analysis of the amino acid sequence of SEQ ID NO:39.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the polypeptide having the aminoacid sequence shown in FIGS. 28A-B (SEQ ID NO:39), which was determinedby sequencing a cloned cDNA. The nucleotide sequence shown in FIGS.28A-B (SEQ ID NO:21) was obtained by sequencing a cloned cDNA (HOUDJ81),which was deposited on Nov. 17, 1998 at the American Type CultureCollection, and given Accession Number 203484. The deposited gene isinserted in the pSport plasmid (Life Technologies, Rockville, Md.) usingthe SalI/NotI restriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO:21 is intended DNA fragments atleast about 15 nt, and more preferably at least about 20 nt; still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:21. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:21. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Representative examples of polynucleotide fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, and from about 501to about 550, and from about 551 to about 600, from about 601 to about650, from about 651 to about 700, from about 701 to about 750, fromabout 751 to about 800, from about 801 to about 850, from about 851 toabout 900, from about 901 to about 950, from about 951 to about 1000,from about 1001 to about 1050, from about 1051 to about 1100, from about1101 to about 1150, from about 1151 to about 1200, from about 1201 toabout 1250, from about 1251 to about 1300, from about 1301 to about1350, from about 1351 to about 1400, from about 1401 to about 1450, fromabout 1451 to about 1500, from about 1501 to about 1520 of SEQ ID NO:21,or the complementary strand thereto, or the cDNA contained in thedeposited gene. In this context “about” includes the particularlyrecited ranges, larger or smaller by several (5, 4, 3, 2, or 1)nucleotides, at either terminus or at both termini. In additionalembodiments, the polynucleotides of the invention encode functionalattributes of the corresponding protein.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions. The data representing the structural or functionalattributes of the protein set forth in FIG. 30 and/or Table X asdescribed above, was generated using the various modules and algorithmsof the DNA*STAR set on default parameters. In a preferred embodiment,the data presented in columns VIII, IX, XIII, and XIV of Table X can beused to determine regions of the protein which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX, XIII, and/or XIV bychoosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

Certain preferred regions in these regards are set out in FIG. 30, butmay, as shown in Table X, be represented or identified by using tabularrepresentations of the data presented in FIG. 30. The DNA*STAR computeralgorithm used to generate FIG. 30 (set on the original defaultparameters) was used to present the data in FIG. 30 in a tabular format(See Table X). The tabular format of the data in FIG. 30 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 30 and in Table Xinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIGS.28A-B. As set out in FIG. 30 and in Table X such preferred regionsinclude Gamier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, etc.) may still beretained. For example, the ability of shortened muteins to induce and/orbind to antibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a mutein with a large numberof deleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact peptides composed of as few as six aminoacid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence shown in FIGS. 28A-B, up to the threonine residue at positionnumber 359 and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues n1-364 of FIGS. 28A-B, where n1 is aninteger from 2 to 359 corresponding to the position of the amino acidresidue in FIGS. 28A-B (which is identical to the sequence shown as SEQID NO:39). N-terminal deletions of the polypeptide of the inventionshown as SEQ ID NO:39 include polypeptides comprising the amino acidsequence of residues: A-2 to R-364; S-3 to R-364; V-4 to R-364; V-5 toR-364; L-6 to R-364; P-7 to R-364; S-8 to R-364; G-9 to R-364; S-10 toR-364; Q-11 to R-364; C-12 to R-364; A-13 to R-364; A-14 to R-364; A-15to R-364; A-16 to R-364; A-17 to R-364; A-18 to R-364; A-19 to R-364;A-20 to R-364; P-21 to R-364; P-22 to R-364; G-23 to R-364; L-24 toR-364; R-25 to R-364; L-26 to R-364; R-27 to R-364; L-28 to R-364; L-29to R-364; L-30 to R-364; L-31 to R-364; L-32 to R-364; F-33 to R-364;S-34 to R-364; A-35 to R-364; A-36 to R-364; A-37 to R-364; L-38 toR-364; I-39 to R-364; P-40 to R-364; T-41 to R-364; G-42 to R-364; D-43to R-364; G-44 to R-364; Q-45 to R-364; N-46 to R-364; L-47 to R-364;F-48 to R-364; T-49 to R-364; K-50 to R-364; D-51 to R-364; V-52 toR-364; T-53 to R-364; V-54 to R-364; I-55 to R-364; E-56 to R-364; G-57to R-364; E-58 to R-364; V-59 to R-364; A-60 to R-364; T-61 to R-364;I-62 to R-364; S-63 to R-364; C-64 to R-364; Q-65 to R-364; V-66 toR-364; N-67 to R-364; K-68 to R-364; S-69 to R-364; D-70 to R-364; D-71to R-364; S-72 to R-364; V-73 to R-364; I-74 to R-364; Q-75 to R-364;L-76 to R-364; L-77 to R-364; N-78 to R-364; P-79 to R-364; N-80 toR-364; R-81 to R-364; Q-82 to R-364; T-83 to R-364; I-84 to R-364; Y-85to R-364; F-86 to R-364; R-87 to R-364; D-88 to R-364; F-89 to R-364;R-90 to R-364; P-91 to R-364; L-92 to R-364; K-93 to R-364; D-94 toR-364; S-95 to R-364; R-96 to R-364; F-97 to R-364; Q-98 to R-364; L-99to R-364; L-100 to R-364; N-101 to R-364; F-102 to R-364; S-103 toR-364; S-104 to R-364; S-105 to R-364; E-106 to R-364; L-107 to R-364;K-108 to R-364; V-109 to R-364; S-110 to R-364; L-111 to R-364; T-112 toR-364; N-113 to R-364; V-114 to R-364; S-115 to R-364; I-116 to R-364;S-117 to R-364; D-118 to R-364; E-119 to R-364; G-120 to R-364; R-121 toR-364; Y-122 to R-364; F-123 to R-364; C-124 to R-364; Q-125 to R-364;L-126 to R-364; Y-127 to R-364; T-128 to R-364; D-129 to R-364; P-130 toR-364; P-131 to R-364; Q-132 to R-364; E-133 to R-364; S-134 to R-364;Y-135 to R-364; T-136 to R-364; T-137 to R-364; I-138 to R-364; T-139 toR-364; V-140 to R-364; L-141 to R-364; V-142 to R-364; P-143 to R-364;P-144 to R-364; R-145 to R-364; N-146 to R-364; L-147 to R-364; M-148 toR-364; I-149 to R-364; D-150 to R-364; I-151 to R-364; Q-152 to R-364;K-153 to R-364; D-154 to R-364; T-155 to R-364; A-156 to R-364; V-157 toR-364; E-158 to R-364; G-159 to R-364; E-160 to R-364; E-161 to R-364;I-162 to R-364; E-163 to R-364; V-164 to R-364; N-165 to R-364; C-166 toR-364; T-167 to R-364; A-168 to R-364; M-169 to R-364; A-170 to R-364;S-171 to R-364; K-172 to R-364; P-173 to R-364; A-174 to R-364; T-175 toR-364; T-176 to R-364; I-177 to R-364; R-178 to R-364; W-179 to R-364;F-180 to R-364; K-181 to R-364; G-182 to R-364; N-183 to R-364; T-184 toR-364; E-185 to R-364; L-186 to R-364; K-187 to R-364; G-188 to R-364;K-189 to R-364; S-190 to R-364; E-191 to R-364; V-192 to R-364; E-193 toR-364; E-194 to R-364; W-195 to R-364; S-196 to R-364; D-197 to R-364;M-198 to R-364; Y-199 to R-364; T-200 to R-364; V-201 to R-364; T-202 toR-364; S-203 to R-364; Q-204 to R-364; L-205 to R-364; M-206 to R-364;L-207 to R-364; K-208 to R-364; V-209 to R-364; H-210 to R-364; K-211 toR-364; E-212 to R-364; D-213 to R-364; D-214 to R-364; G-215 to R-364;V-216 to R-364; P-217 to R-364; V-218 to R-364; I-219 to R-364; C-220 toR-364; Q-221 to R-364; V-222 to R-364; E-223 to R-364; H-224 to R-364;P-225 to R-364; A-226 to R-364; V-227 to R-364; T-228 to R-364; G-229 toR-364; N-230 to R-364; L-231 to R-364; Q-232 to R-364; T-233 to R-364;Q-234 to R-364; R-235 to R-364; Y-236 to R-364; L-237 to R-364; E-238 toR-364; V-239 to R-364; Q-240 to R-364; Y-241 to R-364; K-242 to R-364;P-243 to R-364; Q-244 to R-364; V-245 to R-364; H-246 to R-364; I-247 toR-364; Q-248 to R-364; M-249 to R-364; T-250 to R-364; Y-251 to R-364;P-252 to R-364; L-253 to R-364; Q-254 to R-364; G-255 to R-364; L-256.toR-364; T-257 to R-364; R-258 to R-364; E-259 to R-364; G-260 to R-364;D-261 to R-364; A-262 to R-364; L-263 to R-364; E-264 to R-364; L-265 toR-364; T-266 to R-364; C-267 to R-364; E-268 to R-364; A-269 to R-364;I-270 to R-364; G-271 to R-364; K-272 to R-364; P-273 to R-364; Q-274 toR-364; P-275 to R-364; V-276 to R-364; M-277 to R-364; V-278 to R-364;T-279 to R-364; W-280 to R-364; V-281 to R-364; R-282 to R-364; V-283 toR-364; D-284 to R-364; D-285 to R-364; E-286 to R-364; M-287 to R-364;P-288 to R-364; Q-289 to R-364; H-290 to R-364; A-291 to R-364; V-292 toR-364; L-293 to R-364; S-294 to R-364; G-295 to R-364; P-296 to R-364;N-297 to R-364; L-298 to R-364; F-299 to R-364; I-300 to R-364; N-301 toR-364; N-302 to R-364; L-303 to R-364; N-304 to R-364; K-305 to R-364;T-306 to R-364; D-307 to R-364; N-308 to R-364; G-309 to R-364; T-310 toR-364; Y-311 to R-364; R-312 to R-364; C-313 to R-364; E-314 to R-364;A-315 to R-364; S-316 to R-364; N-317 to R-364; I-318 to R-364; V-319 toR-364; G-320 to R-364; K-321 to R-364; A-322 to R-364; H-323 to R-364;S-324 to R-364; D-325 to R-364; Y-326 to R-364; M-327 to R-364; L-328 toR-364; Y-329 to R-364; V-330 to R-364; Y-331 to R-364; D-332 to R-364;P-333 to R-364; P-334 to R-364; T-335 to R-364; T-336 to R-364; I-337 toR-364; P-338 to R-364; P-339 to R-364; P-340 to R-364; T-341 to R-364;T-342 to R-364; T-343 to R-364; T-344 to R-364; T-345 to R-364; T-346 toR-364; T-347 to R-364; T-348 to R-364; T-349 to R-364; T-350 to R-364;T-351 to R-364; T-352 to R-364; T-353 to R-364; 1-354 to R-364; L-355 toR-364; T-356 to R-364; 1-357 to R-364; 1-358 to R-364; T-359 to R-364;of SEQ ID NO:39. Polynucleotides encoding these polypeptides are alsoencompassed by the invention, as are antibodies that bind one or more ofthese polypeptides. Moreover, fragments and variants of thesepolypeptides (e.g., fragments as described herein, polypeptides at least80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to thesepolypeptides and polypeptides encoded by the polynucleotide whichhybridizes, under stringent conditions, to the polynucleotide encodingthese polypeptides, or the complement thereof) are encompassed by theinvention. Antibodies that bind these fragments and variants of theinvention are also encompassed by the invention. Polynucleotidesencoding these fragments and variants are also encompassed by theinvention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities such as ability to modulate theextracellular matrix, etc.) may still be retained. For example theability to induce and/or bind to antibodies which recognize the completeor mature forms of the polypeptide generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the C-terminus. Whether a particular polypeptidelacking C-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata mutein with a large number of deleted C-terminal amino acid residuesmay retain some biological or immunogenic activities. In fact, peptidescomposed of as few as six amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the polypeptide shown in FIGS. 28A-B, up to the leucineresidue at position number 6, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues 1-m1of FIGS. 28A-B, wherem1 integer from 6 to 364 corresponding to the position of the amino acidresidue in FIGS. 28A-B. Moreover, the invention provides polynucleotidesencoding polypeptides comprising, or alternatively consisting of, theamino acid sequence of C-terminal deletions of the polypeptide of theinvention shown as SEQ ID NO:39 include polypeptides comprising theamino acid sequence of residues: M-1 to A-363; M-1 to R-362; M-1 toS-361; M-1 to D-360; M-1 to T-359; M-1 to I-358; M-1 to I-357; M-1 toT-356; M-1 to L-355; M-1 to I-354; M-1 to T-353; M-1 to T-352; M-1 toT-351; M-1 to T-350; M-1 to T-349; M-1 to T-348; M-1 to T-347; M-1 toT-346; M-1 to T-345; M-1 to T-344; M-1 to T-343; M-1 to T-342; M-1 toT-341; M-1 to P-340; M-1 to P-339; M-1 to P-338; M-1 to I-337; M-1 toT-336; M-1 to T-335; M-1 to P-334; M-1 to P-333; M-1 to D-332; M-1 toY-331; M-1 to V-330; M-1 to Y-329; M-1 to L-328; M-1 to M-327; M-1 toY-326; M-1 to D-325; M-1 to S-324; M-1 to H-323; M-1 to A-322; M-1 toK-321; M-1 to G-320; M-I to V-319; M-1 to I-318; M-1 to N-317; M-1 toS-316; M-1 to A-315; M-1 to E-314; M-1 to C-313; M-1 to R-312; M-1 toY-311; M-1 to T-310; M-1 to G-309; M-1 to N-308; M-1 to D-307; M-1 toT-306; M-1 to K-305; M-1 to N-304;M-1 to L-303; M-1 to N-302; M-1 toN-301; M-1 to I-300; M-1 to F-299; M-1 to L-298; M-1 to N-297; M-1 toP-296; M-1 to G-295; M-1 to S-294; M-1 to L-293; M-1 to V-292; M-1 toA-291; M-1 to H-290; M-1 to Q-289; M-1 to P-288; M-1 to M-287; M-1 toE-286; M-1 to D-285; M-1 to D-284; M-1 to V-283; M-1 to R-282; M-1 toV-281; M-1 to W-280; M-1 to T-279; M-1 to V-278; M-1 to M-277; M-1 toV-276; M-1 to P-275; M-1 to Q-274; M-1 to P-273; M-1 to K-272; M-1 toG-271; M-1 to I-270; M-1 to A-269; M-1 to E-268; M-1 to C-267; M-1 toT-266; M-1 to L-265; M-1 to E-264; M-1 to L-263; M-1 to A-262; M-1 toD-261; M-1 to G-260; M-1 to E-259; M-1 to R-258; M-1 to T-257; M-1 toL-256; M-1 to G-255; M-1 to Q-254; M-1 to L-253; M-1 to P-252; M-1 toY-251; M-1 to T-250; M-1 to M-249; M-1 to Q-248; M-1 to I-247; M-1 toH-246; M-1 to V-245; M-1 to Q-244; M-1 to P-243; M-1 to K-242; M-1 toY-241; M-1 to Q-240; M-1 to V-239; M-1 to E-238; M-1 to L-237; M-1 toY-236; M-1 to R-235; M-1 to Q-234; M-1 to T-233; M-1 to Q-232; M-1 toL-231; M-1 to N-230; M-1 to G-229; M-1 to T-228; M-1 to V-227; M-1 toA-226; M-1 to P-225; M-1 to H-224; M-1 to E-223; M-1 to V-222; M-1 toQ-221; M-1 to C-220; M-1 to 1-219; M-1 to V-218; M-1 to P-217; M-1 toV-216; M-1 to G-215; M-1 to D-214; M-1 to D-213; M-1 to E-212; M-1 toK-211; M-1 to H-210; M-1 to V-209; M-1 to K-208; M-1 to L-207; M-1 toM-206; M-1 to L-205; M-1 to Q-204; M-1 to S-203; M-1 to T-202; M-1 toV-201; M-1 to T-200; M-1 to Y-199; M-1 to M-198; M-1 to D-197; M-1 toS-196; M-1 to W-195; M-1 to E-194; M-1 to E-193; M-1 to V-192; M-1 toE-191; M-1 to S-190; M-1 to K-189; M-1 to G-188; M-1 to K-187; M-1 toL-186; M-1 to E-185; M-1 to T-184; M-1 to N-183; M-1 to G-1 82; M-1 toK-181; M-1 to F-180; M-1 to W-179; M-1 to R-178; M-1 to 1-177; M-1 toT-176; M-1 to T-175; M-1 to A-174; M-1 to P-173; M-1 to K-172; M-1 toS-1 71; M-1 to A-1 70; M-1 to M-169; M-1 to A-168; M-1 to T-167; M-1 toC-166; M-1 to N-165; M-1 to V-164; M-1 to E-163; M-1 to I-162; M-1 toE-161; M-1 to E-160; M-1 to G-159; M-1 to E-158; M-1 to V-157; M-1 toA-156; M-1 to T-155; M-1 to D-154; M-1 to K-1 53; M-1 to Q-152; M-1 toI-151; M-1 to D-150; M-1 to 1-149; M-1 to M-148; M-1 to L-147; M-1 toN-146; M-1 to R-145; M-1 to P-144; M-1 to P-143; M-1 to V-142; M-1 toL-141; M-1 to V-140; M-1 to T-139; M-1 to I-138; M-1 to T-137; M-1 toT-136; M-1 to Y-135; M-1 to S-134; M-1 to E-133; M-1 to Q-132; M-1 toP-131; M-1 to P-130; M-1 to D-129; M-1 to T-128; M-1 to Y-127; M-1 toL-126; M-1 to Q-125; M-1 to C-124; M-1 to F-123; M-1 to Y-122; M-1 toR-121; M-1 to G-120; M-1 to E-119; M-1 to D-118; M-1 to S-117; M-1 toI-116; M-1 to S-115; M-1 to V-114; M-1 to N-113; M-1 to T-112; M-1 toL-111; M-1 to S-110; M-1 to V-109; M-1 to K-108; M-1 to L-107; M-1 toE-106; M-1 to S-105; M-1 to S-104; M-1 to S-103; M-1 to F-102; M-1 toN-101; M-1 to L-100; M-1 to L-99; M-1 to Q-98; M-1 to F-97; M-1 to R-96;M-1 to S-95; M-1 to D-94; M-1 to K-93; M-1 to L-92; M-1 to P-91; M-1 toR-90; M-1 to F-89; M-1 to D-88; M-1 to R-87; M-1 to F-86; M-1 to Y-85;M-1 to I-84; M-1 to T-83; M-1 to Q-82; M-1 to R-81; M-1 to N-80; M-1 toP-79; M-1 to N-78; M-1 to L-77; M-1 to L-76; M-1 to Q-75; M-1 to I-74;M-1 to V-73; M-1 to S-72; M-1 to D-71; M-1 to D-70; M-1 to S-69; M-1 toK-68; M-1 to N-67; M-1 to V-66; M-1 to Q-65; M-1 to C-64; M-1 to S-63;M-1 to I-62; M-1 to T-61; M-1 to A-60; M-1 to V-59; M-1 to E-58; M-1 toG-57; M-1 to E-56; M-1 to I-55; M-1 to V-54; M-1 to T-53; M-1 to V-52;M-1 to D-51; M-1 to K-50; M-1 to T-49; M-1 to F-48; M-1 to L-47; M-1 toN-46; M-1 to Q-45; M-1 to G-44; M-1 to D-43; M-1 to G-42; M-1 to T-41;M-1 to P-40; M-1 to I-39; M-1 to L-38; M-1 to A-37; M-1 to A-36; M-1 toA-35; M-1 to S-34; M-1 to F-33; M-1 to L-32; M-1 to L-31; M-1 to L-30;M-1 to L-29; M-1 to L-28; M-1 to R-27; M-1 to L-26; M-1 to R-25; M-1 toL-24; M-1 to G-23; M-1 to P-22; M-1 to P-21; M-1 to A-20; M-1 to A-19;M-1 to A-18; M-1 to A-17; M-1 to A-16; M-1 to A-15; M-1 to A-14; M-1 toA-13; M-1 to C-12; M-1 to Q-11; M-1 to S-10; M-1 to G-9; M-1 to S-8; M-1to P-7; M-1 to L-6; of SEQ ID NO:39. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g., fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:21 whichhave been determined from the following related cDNA genes: HSQFJ92R(SEQ ID NO:139), HFLAB18F (SEQ ID NO:140), HAQBH82R (SEQ ID NO:141),HLHTM10R (SEQ ID NO:142), and HLHAL65R (SEQ ID NO: 143).

This gene is expressed primarily in immune system related tissues suchas ulcerative colitis, rejected kidney tissues, and to a lesser extentin thymus and bone marrow.

Therefore, polynucleotides and polypeptides of the invention, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of diseases and conditions which include, but are not limitedto, immune and hematopoietic diseases and/or disorders, particularlyulcerative colitis and rejected organs. Similarly, polypeptides andantibodies directed to these polypeptides are useful in providingimmunological probes for differential identification of the tissue(s) orcell type(s). For a number of disorders of the above tissues or cells,particularly of the immune system, expression of this gene atsignificantly higher or lower levels is routinely detected in certaintissues or cell types (e.g. transplanted kidney, immune, hematopoeitic,renal, and cancerous and wounded tissues) or bodily fluids (e.g., lymph,serum, plasma, urine, synovial fluid and spinal fluid) or another tissueor cell sample taken from an individual having such a disorder, relativeto the standard gene expression level, i.e., the expression level inhealthy tissue or bodily fluid from an individual not having thedisorder.

The tissue distribution primarily in immune cells and tissues, combinedwith the homology to the CRTAM, thymocyte activation and developmentalprotein, the class-l MHC-restricted T cell associated molecule protein,and the polivirus receptor, indicates that the protein products of thisgene are useful for the regulation of celluar physiology, development,differentiation or function of various cell types, includinghaematopoietic cells and particularly T-cell progenitors. Representativeuses are described in the “Immune Activity” and “infectious disease”sections below, in Example 11, 13, 14, 16, 18, 19, 20, and 27, andelsewhere herein. The proteins can be used to develop products for thediagnosis and treatment of conditions associated with abnormalphysiology or development, including abnormal proliferation, e.g.cancers, or degenerative conditions. The physiology or development of acell can be modulated by contacting the cell with an agonist orantagonist (i.e. an anti-CRTAM-like peptide antibody). Further theCRTAM-like polypeptides of the present invention include treatment ofulcerative colitis, organ rejection and other immune system relateddisorders. Agonists or antagonists may treat or prevent such disordersas ulcerative colitis and rejected organs, such as kidney. Based uponthe tissue distribution of this protein, antagonists directed againstthis protein is useful in blocking the activity of this protein.Accordingly, preferred are antibodies which specifically bind a portionof the translation product of this gene.

Also provided is a kit for detecting tumors in which expression of thisprotein occurs. Such a kit comprises in one embodiment an antibodyspecific for the translation product of this gene bound to a solidsupport. Also provided is a method of detecting these tumors in anindividual which comprises a step of contacting an antibody specific forthe translation product of this gene to a bodily fluid from theindividual, preferably serum, and ascertaining whether antibody binds toan antigen found in the bodily fluid. Preferably the antibody is boundto a solid support and the bodily fluid is serum. The above embodiments,as well as other treatments and diagnostic tests (kits and methods), aremore particularly described elsewhere herein. Furthermore, the proteinmay also be used to determine biological activity, to raise antibodies,as tissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:21 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 1506 of SEQID NO:21, b is an integer of 15 to 1520, where both a and b correspondto the positions of nucleotide residues shown in SEQ ID NO:21, and whereb is greater than or equal to a+14.

Features of Protein Encoded by Gene No: 12

FIG. 31 shows the nucleotide (SEQ ID NO:22) and deduced amino acidsequence (SEQ ID NO:40) of the present invention. Predicted amino acidsfrom about 1 to about 23 constitute the predicted signal peptide (aminoacid residues from about 1 to about 23 in SEQ ID NO:40) and arerepresented by the underlined amino acid regions.

FIG. 32 shows the regions of similarity between the amino acid sequencesof the present invention SEQ ID NO:40 and the human FAP protein(gi|1890647) (SEQ ID NO:146).

FIG. 33 shows an analysis of the amino acid sequence of SEQ ID NO:40.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the polypeptide having the aminoacid sequence shown in FIG. 31 (SEQ ID NO:40), which was determined bysequencing a cloned cDNA. The nucleotide sequence shown in FIG. 31 (SEQID NO:22) was obtained by sequencing a cloned cDNA (HPWCM76), which wasdeposited on Nov. 17, 1998 at the American Type Culture Collection, andgiven Accession Number 203484. The deposited gene is inserted in thepSport plasmid (Life Technologies, Rockville, Md.) using the SalI/NotIrestriction endonuclease cleavage sites.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatedDNA molecule having the nucleotide sequence of the deposited cDNA or thenucleotide sequence shown in SEQ ID NO:22 is intended DNA fragments atleast about 15 nt, and more preferably at least about 20 nt, still morepreferably at least about 30 nt, and even more preferably, at leastabout 40 nt in length which are useful as diagnostic probes and primersas discussed herein. Of course, larger fragments 50-1500 nt in lengthare also useful according to the present invention, as are fragmentscorresponding to most, if not all, of the nucleotide sequence of thedeposited cDNA or as shown in SEQ ID NO:22. By a fragment at least 20 ntin length, for example, is intended fragments which include 20 or morecontiguous bases from the nucleotide sequence of the deposited cDNA orthe nucleotide sequence as shown in SEQ ID NO:22. In this context“about” includes the particularly recited size, larger or smaller byseveral (5, 4, 3, 2, or 1) nucleotides, at either terminus or at bothtermini. Representative examples of polynucleotide fragments of theinvention include, for example, fragments that comprise, oralternatively, consist of, a sequence from about nucleotide 1 to about50, from about 51 to about 100, from about 101 to about 150, from about151 to about 200, from about 201 to about 250, from about 251 to about300, from about 301 to about 350, from about 351 to about 400, fromabout 401 to about 450, from about 451 to about 500, and from about 501to about 550, and from about 551 to about 600, from about 601 to about650, from about 651 to about 700, from about 701 to about 750, fromabout 751 to about 800, from about 801 to about 807 of SEQ ID NO:22, orthe complementary strand thereto, or the cDNA contained in the depositedgene. In this context “about” includes the particularly recited ranges,larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. In additional embodiments, thepolynucleotides of the invention encode functional attributes of thecorresponding protein.

Preferred embodiments of the invention in this regard include fragmentsthat comprise alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, flexible regions, surface-forming regions and high antigenicindex regions. The data representing the structural or functionalattributes of the protein set forth in FIG. 33 and/or Table XI, asdescribed above, was generated using the various modules and algorithmsof the DNA*STAR set on default parameters. In a preferred embodiment,the data presented in columns VIII, IX, XIII, and XIV of Table XI can beused to determine regions of the protein which exhibit a high degree ofpotential for antigenicity. Regions of high antigenicity are determinedfrom the data presented in columns VIII, IX, XIII, and/or XIV bychoosing values which represent regions of the polypeptide which arelikely to be exposed on the surface of the polypeptide in an environmentin which antigen recognition may occur in the process of initiation ofan immune response.

Certain preferred regions in these regards are set out in FIG. 33, butmay, as shown in Table XI, be represented or identified by using tabularrepresentations of the data presented in FIG. 33. The DNA*STAR computeralgorithm used to generate FIG. 33 (set on the original defaultparameters) was used to present the data in FIG. 33 in a tabular format(See Table XI). The tabular format of the data in FIG. 33 is used toeasily determine specific boundaries of a preferred region. Theabove-mentioned preferred regions set out in FIG. 33 and in Table XIinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIG. 31. Asset out in FIG. 33 and in Table XI, such preferred regions includeGarnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions,Kyte-Doolittle hydrophilic regions and Hopp-Woods hydrophobic regions,Eisenberg alpha- and beta-amphipathic regions, Karplus-Schulz flexibleregions, Jameson-Wolf regions of high antigenic index and Eminisurface-forming regions. Even if deletion of one or more amino acidsfrom the N-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, etc.) may still beretained. For example, the ability of shortened muteins to induce and/orbind to antibodies which recognize the complete or mature forms of thepolypeptides generally will be retained when less than the majority ofthe residues of the complete or mature polypeptide are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete polypeptide retains such immunologic activities canreadily be determined by routine methods described herein and otherwiseknown in the art. It is not unlikely that a mutein with a large numberof deleted N-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixamino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence shown in FIG. 31, up to the arginine residue at position number61 and polynucleotides encoding such polypeptides. In particular, thepresent invention provides polypeptides comprising the amino acidsequence of residues n1-66 of FIG. 31, where n1 is an integer from 2 to61 corresponding to the position of the amino acid residue in FIG. 31(which is identical to the sequence shown as SEQ ID NO:40). N-terminaldeletions of the polypeptide of the invention shown as SEQ ID NO:40include polypeptides comprising the amino acid sequence of residues: S-2to N-66; S-3 to N-66; S-4 to N-66; S-5 to N-66; L-6 to N-66; K-7 toN-66; H-8 to N-66; L-9 to N-66; L-10 to N-66; C-11 to N-66; M-12 toN-66; A-13 to N-66; L-14 to N-66; S-15 to N-66; W-16 to N-66; F-17 toN-66; S-18 to N-66; S-19 to N-66; F-20 to N-66; I-21 to N-66; S-22 toN-66; G-23 to N-66; E-24 to N-66; T-25 to N-66; S-26 to N-66; F-27 toN-66; S-28 to N-66; L-29 to N-66; L-30 to N-66; N-31 to N-66; S-32 toN-66; F-33 to N-66; F-34 to N-66; L-35 to N-66; P-36 to N-66; Y-37 toN-66; P-38 to N-66; S-39 to N-66; S-40 to N-66; R-41 to N-66; C-42 toN-66; C-43 to N-66; C-44 to N-66; F-45 to N-66; S-46 to N-66; V-47 toN-66; Q-48 to N-66; C-49 to N-66; S-50 to N-66; I-51 to N-66; L-52 toN-66; D-53 to N-66; P-54 to N-66; F-55 to N-66; S-56 to N-66; C-57 toN-66; N-58 to N-66; S-59 to N-66; M-60 to N-66; R-61 to N-66; of SEQ IDNO:40. Polynucleotides encoding these polypeptides are also encompassedby the invention, as are antibodies that bind one or more of thesepolypeptides. Moreover, fragments and variants of these polypeptides(e.g., fragments as described herein, polypeptides at least 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to these polypeptides andpolypeptides encoded by the polynucleotide which hybridizes, understringent conditions, to the polynucleotide encoding these polypeptides,or the complement thereof) are encompassed by the invention. Antibodiesthat bind these fragments and variants of the invention are alsoencompassed by the invention. Polynucleotides encoding these fragmentsand variants are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification or loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities such as ability to modulate theextracellular matrix, etc.) may still be retained. For example theability to induce and/or bind to antibodies which recognize the completeor mature forms of the polypeptide generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the C-terminus. Whether a particular polypeptidelacking C-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thata mutein with a large number of deleted C-terminal amino acid residuesmay retain some biological or immunogenic activities. In fact, peptidescomposed of as few as six amino acid residues may often evoke an immuneresponse.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the polypeptide shown in FIG. 31, up to the leucine residueat position number 6, and polynucleotides encoding such polypeptides. Inparticular, the present invention provides polypeptides comprising theamino acid sequence of residues 1-ml of FIG. 31, where ml is an integerfrom 6 to 66 corresponding to the position of the amino acid residue inFIG. 31. Moreover, the invention provides polynucleotides encodingpolypeptides comprising, or alternatively consisting of, the amino acidsequence of C-terminal deletions of the polypeptide of the inventionshown as SEQ ID NO:40 include polypeptides comprising the amino acidsequence of residues: M-1 to E-65; M-1 to W-64; M-1 to P-63; M-1 toF-62; M-1 to R-61; M-1 to M-60; M-1 to S-59; M-1 to N-58; M-1 to C-57;M-1 to S-56; M-1 to F-55; M-1 to P-54; M-1 to D-53; M-1 to L-52; M-1 toI-51; M-1 to S-50; M-1 to C-49; M-1 to Q-48; M-1 to V-47; M-1 to S-46;M-1 to F-45; M-1 to C-44; M-1 to C-43; M-1 to C-42; M-1 to R-41; M-1 toS-40; M-1 to S-39; M-1 to P-38; M-1 to Y-37; M-1 to P-36; M-1 to L-35;M-1 to F-34; M-1 to F-33; M-1 to S-32; M-1 to N-31; M-1 to L-30; M-1 toL-29; M-1 to S-28; M-1 to F-27; M-1 to S-26; M-1 to T-25; M-1 to E-24;M-1 to G-23; M-1 to S-22; M-1 to I-21; M-1 to F-20; M-1 to S-19; M-1 toS-18; M-1 to F-17; M-1 to W-16; M-1 to S-15; M-1 to L-14; M-1 to A-13;M-1 to M-12; M-1 to C-11; M-1 to L-10; M-1 to L-9; M-1 to H-8; M-1 toK-7; M-1 to L-6; of SEQ ID NO:40. Polynucleotides encoding thesepolypeptides are also encompassed by the invention, as are antibodiesthat bind one or more of these polypeptides. Moreover, fragments andvariants of these polypeptides (e.g., fragments as described herein,polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%,identical to these polypeptides and polypeptides encoded by thepolynucleotide which hybridizes, under stringent conditions, to thepolynucleotide encoding these polypeptides, or the complement thereof)are encompassed by the invention. Antibodies that bind these fragmentsand variants of the invention are also encompassed by the invention.Polynucleotides encoding these fragments and variants are alsoencompassed by the invention.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:22 whichhave been determined from the following related cDNA genes: HPWCM76R(SEQ ID NO: 147).

This gene is expressed primarily in prostate BPH (benign prostatichyperplasia) tissue.

Therefore, polynucleotides and polypeptides of the invention, includingantibodies, are useful as reagents for differential identification ofthe tissue(s) or cell type(s) present in a biological sample and fordiagnosis of diseases and conditions which include, but are not limitedto, inflammation of the prostate, or related tissues. Similarly,polypeptides and antibodies directed to these polypeptides are useful inproviding immunological probes for differential identification of thetissue(s) or cell type(s). For a number of disorders of the abovetissues or cells, particularly of the prostate, expression of this geneat significantly higher or lower levels is routinely detected in certaintissues or cell types (e.g. prostate, cancerous and wounded tissues) orbodily fluids (e.g., serum, plasma, urine, synovial fluid and spinalfluid) or another tissue or cell sample taken from an individual havingsuch a disorder, relative to the standard gene expression level, i.e.,the expression level in healthy tissue or bodily fluid from anindividual not having the disorder.

The tissue distribution in prostate BPH tissue indicates thatpolynucleotides and polypeptides corresponding to this gene are usefulfor the treatment of inflammatory conditions which result in anenlargement of the prostate, or related tissues. Polynucleotides andpolypeptides corresponding to this gene are useful for the treatment anddiagnosis of conditions concerning proper testicular function (e.g.endocrine function, sperm maturation), as well as cancer. Therefore,this gene product is useful in the treatment of male infertility and/orimpotence. This gene product is also useful in assays designed toidentify binding agents, as such agents (antagonists) are useful as malecontraceptive agents. Similarly, the protein is believed to be useful inthe treatment and/or diagnosis of testicular cancer. The testes are alsoa site of active gene expression of transcripts that is expressed,particularly at low levels, in other tissues of the body. Therefore,this gene product is expressed in other specific tissues or organs whereit may play related functional roles in other processes, such ashematopoiesis, inflammation, bone formation, and kidney function, toname a few possible target indications. Based upon the tissuedistribution of this protein, antagonists directed against this proteinis useful in blocking the activity of this protein. Accordingly,preferred are antibodies which specifically bind a portion of thetranslation product of this gene.

Also provided is a kit for detecting tumors in which expression of thisprotein occurs. Such a kit comprises in one embodiment an antibodyspecific for the translation product of this gene bound to a solidsupport. Also provided is a method of detecting these tumors in anindividual which comprises a step of contacting an antibody specific forthe translation product of this gene to a bodily fluid from theindividual, preferably serum, and ascertaining whether antibody binds toan antigen found in the bodily fluid. Preferably the antibody is boundto a solid support and the bodily fluid is serum. The above embodiments,as well as other treatments and diagnostic tests (kits and methods), aremore particularly described elsewhere herein.

Shared homology to the FAP protein indicates that the protein product ofthis gene is useful in treating, detecting, and/or preventing ironmetabolism disorders, particularly those resulting in high oxidativestates, tissue damage, athersclerosis, free radical damage, vasculardisorders, iron binding protein dysfunction, nitric oxide synthasedysfunction or aberration, vasodilation disorders, and tissue edema.Based on the sequence similarity, the translation product of this geneis expected to share at least some biological activities with ironmetabolism modulatory proteins. Such activities are known in the art,some of which are described elsewhere herein. Furthermore, the proteinmay also be used to determine biological activity, to raise antibodies,as tissue markers, to isolate cognate ligands or receptors, to identifyagents that modulate their interactions, in addition to its use as anutritional supplement. Protein, as well as, antibodies directed againstthe protein may show utility as a tumor marker and/or immunotherapytargets for the above listed tissues.

Many polynucleotide sequences, such as EST sequences, are publiclyavailable and accessible through sequence databases. Some of thesesequences are related to SEQ ID NO:22 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. To list every related sequence is cumbersome.Accordingly, preferably excluded from the present invention are one ormore polynucleotides comprising a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 793 of SEQID NO:22, b is an integer of 15 to 807, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:22, and where bis greater than or equal to a +14.

TABLE I Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . B .. . . −0.10 0.44 . . . −0.40 0.48 Val 2 . . B . . . . 0.08 0.01 . . .0.15 0.63 Ala 3 . . B . . . . 0.47 0.01 . . . 0.40 0.76 Gln 4 . . B . .. . 0.51 −0.01 . . . 1.40 1.33 Asp 5 . . . . . T C 0.23 −0.20 . . F 2.201.77 Pro 6 . . . . T T . 0.02 −0.27 * * F 2.50 0.94 Gln 7 . . . . T T .0.88 −0.09 * * F 2.25 0.45 Gly 8 . . . . T T . 0.66 −0.09 . * F 2.000.47 Cys 9 . A B . . . . −0.01 0.60 . * . −0.10 0.25 Leu 10 . A B . . .. −0.82 0.74 . * . −0.35 0.08 Gln 11 . A B . . . . −0.91 1.03 . * .−0.60 0.06 Leu 12 . A B . . . . −0.91 0.99 . * . −0.60 0.16 Cys 13 . A B. . . . −1.42 0.41 . * . −0.60 0.34 Leu 14 . A B . . . . −1.34 0.37 * .. −0.30 0.14 Ser 15 . A B . . . . −0.53 0.47 * * . −0.60 0.18 Glu 16 . AB . . . . −0.88 0.19 * . . −0.30 0.53 Val 17 . . B . . T . −0.880.04 * * . 0.10 0.63 Ala 18 A . . . . T . −0.10 0.04 * . . 0.10 0.39 Asn19 . . . . T T . 0.71 −0.34 * . . 1.31 0.44 Gly 20 . . . . T T . 0.800.06 * * F 1.07 0.96 Leu 21 . . . . . . C −0.06 −0.16 * * F 1.63 1.47Arg 22 . . . . . . C 0.50 −0.01 * * F 1.69 0.68 Asn 23 . . . . . T C0.49 −0.03 * . F 2.10 0.92 Pro 24 . . B . . T . −0.37 0.16 * . F 1.241.10 Val 25 . . B . . T . −0.06 0.11 * * . 0.73 0.42 Ser 26 . . B . . T. 0.17 0.61 * * . 0.22 0.35 Met 27 . . B B . . . −0.29 0.71 * . . −0.390.23 Val 28 . . B B . . . −0.29 0.71 . . . −0.60 0.31 His 29 . . B B . .. −0.42 0.07 . . . 0.00 0.38 Ala 30 A . B . . . . 0.12 0.11 . . . 0.500.38 Gly 31 . . . . T T . 0.39 −0.01 * . F 2.15 0.74 Asp 32 . . . . T T. 1.10 −0.16 * * F 2.45 0.74 Gly 33 . . . . . T C 1.26 −0.66 * * F 3.001.44 Thr 34 . . . . . T C 0.59 −0.37 * * F 2.40 1.26 His 35 . . B B . .. 0.32 −0.01 * * . 1.20 0.65 Arg 36 . . B B . . . 0.08 0.63 . * . 0.000.49 Phe 37 . . B B . . . 0.08 0.70 . * . −0.30 0.34 Phe 38 . . B B . .. 0.42 0.21 . * . −0.30 0.44 Val 39 A . . B . . . −0.12 0.11 . * . −0.300.39 Ala 40 A . . B . . . −0.43 0.76 . * . −0.60 0.33 Glu 41 A . . B . .. −1.40 0.40 . * . −0.60 0.38 Gln 42 A . . B . . . −1.56 0.26 . . .−0.30 0.38 Val 43 A . . B . . . −1.14 0.26 . . . −0.30 0.28 Gly 44 . . BB . . . −1.14 0.67 . . . −0.60 0.17 Val 45 . . B B . . . −0.80 1.31 . .. −0.60 0.07 Val 46 . . B B . . . −1.61 1.67 . . . −0.60 0.15 Trp 47 . .B B . . . −1.82 1.71 . . . −0.60 0.13 Val 48 . . B B . . . −0.97 1.71 .. . −0.60 0.27 Tyr 49 . . B . . . . −0.97 1.07 . . . −0.16 0.60 Leu 50 .. B . . T . −0.41 0.86 * . . 0.28 0.56 Pro 51 . . . . T T . 0.560.33 * * F 1.52 1.01 Asp 52 . . . . T T . 0.03 −0.31 . * F 2.36 1.27 Gly53 . . . . . T C 0.89 −0.39 * * F 2.40 1.27 Ser 54 . A . . . . C 1.13−1.07 * . F 2.06 1.42 Arg 55 . A B . . . . 1.73 −1.10 * * F 1.62 1.47Leu 56 . A B . . . . 1.24 −0.67 * . F 1.38 2.30 Glu 57 . A B . . . .0.43 −0.31 * . F 0.84 1.49 Gln 58 . A B . . . . 0.78 −0.01 * * F 0.450.63 Pro 59 A A . . . . . 0.27 −0.01 * * F 0.60 1.27 Phe 60 A A . . . .. 0.20 −0.01 * * . 0.30 0.60 Leu 61 A A . . . . . 1.01 −0.01 * . . 0.300.70 Asp 62 A A . . . . . 0.12 −0.01 * . . 0.30 0.73 Leu 63 A . . B . .. −0.73 0.24 * . . −0.30 0.59 Lys 64 A . . B . . . −1.33 0.10 . . .−0.30 0.53 Asn 65 . . B B . . . −0.94 0.10 . . . −0.30 0.26 Ile 66 . . BB . . . −0.44 0.59 . * . −0.60 0.46 Val 67 . . B B . . . −0.66 0.39 . .. −0.30 0.33 Leu 68 . . B B . . . −0.13 0.81 * . . −0.60 0.32 Thr 69 . .B B . . . −1.07 1.33 . . F −0.45 0.48 Thr 70 . . B B . . . −1.41 1.33 .. F −0.45 0.45 Pro 71 . . B B . . . −0.52 1.11 . . F −0.45 0.54 Trp 72 .. . B T . . 0.33 0.43 . . . −0.20 0.62 Ile 73 . . B B . . . 1.26 −0.06. * . 0.61 0.75 Gly 74 . . B B . . . 1.22 −0.54 . . F 1.37 0.95 Asp 75 .. . . . T C 0.83 −0.54 . . F 2.28 0.89 Glu 76 . . B . . T . 0.23 −0.67 .. F 2.54 1.10 Arg 77 . . . . T T . 0.18 −0.67 . * F 3.10 0.92 Gly 78 . .. . T T . 0.26 −0.67 . . F 2.79 0.54 Phe 79 A . . . . . . 0.01 0.01 . *. 0.83 0.26 Leu 80 A . . . . . . −0.69 0.51 . * . 0.22 0.13 Gly 81 A . .. . . . −0.72 1.30 . * . −0.09 0.12 Leu 82 A . . . . . . −1.04 1.37 * *. −0.40 0.18 Ala 83 A . . . . . . −0.66 1.01 . * . −0.40 0.34 Phe 84 A .. . . . . −0.66 0.33 . * . −0.10 0.70 His 85 A . . . . T . 0.27 0.69 * *. −0.20 0.73 Pro 86 A . . . . T . 0.58 0.00 . * . 0.25 1.42 Lys 87 A . .. . T . 1.39 0.00 . * . 0.56 2.23 Phe 88 A . . . . T . 2.09 −0.39 * * .1.47 2.63 Arg 89 A . . . . . . 2.83 −0.89 * * . 1.88 3.33 His 90 A . . .T T . 2.17 −1.31 * * . 2.79 3.33 Asn 91 . . . . T T . 2.13 −0.53 . * .3.10 3.33 Arg 92 . . . . T T . 1.20 −0.56 . * . 2.79 2.67 Lys 93 . . . .T T . 1.66 0.13 * * . 1.58 1.37 Phe 94 . . . B T . . 1.30 0.39 * * .0.87 1.34 Tyr 95 . . B B . . . 1.03 0.74 . . . −0.14 1.07 Ile 96 . . B B. . . 0.37 1.13 * . . −0.60 0.72 Tyr 97 . . B . . T . −0.56 1.70 * . .−0.20 0.44 Tyr 98 . . B . . T . −0.60 1.60 . * . −0.20 0.23 Ser 99 . . B. . T . 0.14 0.84 . . . −0.20 0.56 Cys 100 A . . . . T . 0.43 0.16 . . .0.10 0.71 Leu 101 A A . . . . . 1.37 −0.60 . . . 0.60 0.91 Asp 102 A A .. . . . 0.76 −1.36 . . F 0.90 1.35 Lys 103 A A . . . . . 1.00 −1.10 . .F 0.90 1.87 Lys 104 A A . . . . . 1.34 −1.67 . . F 0.90 3.94 Lys 105 A A. . . . . 1.12 −2.36 * * F 0.90 4.71 Val 106 A A . . . . . 2.04 −1.67. * F 0.90 1.65 Glu 107 A A . . . . . 1.16 −1.67 . * F 0.90 1.62 Lys 108A A . . . . . 0.81 −0.99 . * F 0.75 0.57 Ile 109 A A . . . . . 0.77−0.60 . * F 0.90 1.02 Arg 110 A A . . . . . 0.12 −1.24 . . . 0.75 1.02Ile 111 A A . . . . . 1.02 −0.63 . * . 0.60 0.51 Ser 112 A A . . . . .0.17 −0.63 . * F 0.90 1.45 Glu 113 A A . . . . . −0.18 −0.67 * * F 0.750.55 Met 114 A A . . . . . 0.82 −0.29 * * F 0.60 1.05 Lys 115 A A . . .. . 0.12 −0.97 * . F 0.90 1.53 Val 116 . A B . . . . 1.01 −0.86 . . F0.75 0.89 Ser 117 A A . . . . . 1.10 −0.86 . * F 0.90 1.51 Arg 118 A A .. . . . 1.10 −1.04 . . F 0.90 1.17 Ala 119 A A . . . . . 1.74 −0.64 * .F 0.90 2.52 Asp 120 A . . . . T . 1.11 −1.29 * . F 1.30 3.77 Pro 121 A .. . . T . 1.97 −1.17 * . F 1.30 1.94 Asn 122 A . . . . T . 1.46 −1.17. * F 1.30 3.21 Lys 123 A . . . . T . 1.39 −0.99 . * F 1.30 1.59 Ala 124A A . . . . . 1.68 −0.99 . * F 0.90 2.05 Asp 125 A A . . . . . 1.68−1.03 * . F 0.90 1.71 Leu 126 A A . . . . . 2.00 −1.43 * * F 0.90 1.48Lys 127 A A . . . . . 1.14 −1.43 * * F 0.90 2.87 Ser 128 A A . . . . .0.21 −1.29 * * F 0.90 1.28 Glu 129 A A . . . . . −0.01 −0.60 * * F 0.901.08 Arg 130 A A . . . . . −0.01 −0.60 * * F 0.75 0.45 Val 131 A A . . .. . −0.09 −0.60 * * . 0.60 0.58 Ile 132 A A . . . . . −0.13 −0.30 * * .0.30 0.23 Leu 133 A A . . . . . 0.17 −0.30 * * . 0.30 0.21 Glu 134 A A .. . . . −0.04 −0.30 * * . 0.30 0.48 Ile 135 A A . . . . . −0.74−0.51 * * . 0.75 1.06 Glu 136 A A . . . . . −0.19 −0.70 * * F 0.90 1.30Glu 137 A A . . . . . 0.70 −1.00 * . F 0.90 1.01 Pro 138 A . . . . T .1.48 −0.60 . * F 1.30 2.31 Ala 139 A . . . . T . 1.48 −0.79 . . F 1.581.82 Ser 140 A . . . . T . 2.02 −0.39 . . F 1.56 1.69 Asn 141 . . . . .T C 1.68 0.04 . . F 1.44 1.08 His 142 . . . . . T C 1.68 0.04 . . F 1.721.06 Asn 143 . . . . T T . 1.08 −0.06 . . F 2.80 1.37 Gly 144 . . . . TT . 0.86 0.24 . . F 1.77 0.70 Gly 145 . . . . T T . 0.46 0.53 . . F 1.190.43 Gln 146 . A B B . . . 0.11 0.81 . . F 0.11 0.23 Leu 147 . A B B . .. −0.67 0.84 . * . −0.32 0.23 Leu 148 . A B B . . . −0.67 1.10 . * .−0.60 0.19 Phe 149 . A B B . . . −0.67 0.67 . * . −0.60 0.18 Gly 150 . .B B . . . −0.57 0.70 . * . −0.60 0.22 Leu 151 . . B . . T . −1.17 0.77. * . −0.20 0.42 Asp 152 . . . . T T . −0.60 0.70 . * . 0.20 0.48 Gly153 . . . . T T . −0.68 0.67 . . . 0.20 0.76 Tyr 154 . . B . . T . −0.680.93 . * . −0.20 0.65 Met 155 . . B B . . . −0.64 1.03 . . . −0.60 0.33Tyr 156 . . B B . . . −0.18 1.51 . * . −0.60 0.49 Ile 157 . . B B . . .−0.18 1.51 . . . −0.60 0.31 Phe 158 . . B B . . . −0.18 0.76 . . . −0.600.52 Thr 159 . . B B . . . −0.28 0.57 . . F −0.45 0.33 Gly 160 . . . . TT . 0.32 0.24 . . F 0.88 0.46 Asp 161 . . . . T T . −0.02 −0.04 . . F1.71 0.93 Gly 162 . . . . . T C 0.52 −0.33 . . F 1.74 0.65 Gly 163 . . .. . T C 1.22 −0.39 . . F 1.97 0.65 Gln 164 . . . . . . C 1.32 −0.81 * .F 2.30 0.65 Ala 165 . . . . . . C 0.97 −0.39 * . F 1.92 1.01 Gly 166 . .B . . . . 0.62 −0.03 . . F 1.34 0.89 Asp 167 . . B . . T . 0.16 −0.03 .. F 1.31 0.51 Pro 168 . . B . . T . −0.20 0.26 . . F 0.48 0.41 Phe 169 .. B . . T . −0.54 0.54 * . . −0.20 0.36 Gly 170 . . B . . T . 0.040.54 * . . −0.20 0.21 Leu 171 . . B . . . . −0.20 0.94 . . . −0.40 0.22Phe 172 . . B . . . . −0.20 1.01 . . . −0.40 0.26 Gly 173 . . B . . . .0.01 0.63 . * . −0.10 0.46 Asn 174 . . . . . . C 0.76 0.60 . * F 0.550.89 Ala 175 . . . . . . C 0.80 −0.09 . . F 1.90 2.05 Gln 176 . . . . .. C 1.31 −0.49 . * F 2.20 2.78 Asn 177 . . . . . T C 1.20 −0.53 . . F3.00 2.32 Lys 178 . . . . T T . 0.73 −0.24 . * F 2.60 1.89 Ser 179 . . B. . T . 0.39 −0.06 . * F 1.75 0.90 Ser 180 . . B . . T . 1.02 −0.03 * *F 1.45 0.55 Leu 181 . . B . . . . 0.17 −0.43 * * F 0.95 0.55 Leu 182 . .B B . . . −0.64 0.21 * . F −0.15 0.31 Gly 183 . . B B . . . −0.580.51 * * F −0.45 0.19 Lys 184 . . B B . . . −1.17 0.13 * * . −0.30 0.45Val 185 . . B B . . . −0.87 0.13 * * . −0.30 0.38 Leu 186 . . B B . . .−0.91 −0.56 * * . 0.60 0.64 Arg 187 . . B B . . . −0.10 −0.34 * * . 0.300.24 Ile 188 . . B B . . . 0.36 0.06 * . . −0.30 0.52 Asp 189 . . B . .T . −0.28 −0.59 * * . 1.15 1.23 Val 190 . . B . . T . 0.23 −0.77 * * .1.00 0.63 Asn 191 . . B . . T . 0.74 −0.34 * * F 0.85 0.89 Arg 192 . . B. . T . 0.60 −0.64 . * F 1.49 0.72 Ala 193 . . . . T . . 1.14 −0.14 * *F 1.88 1.32 Gly 194 . . . . T T . 1.19 −0.36 * . F 2.27 0.81 Ser 195 . .. . . T C 2.16 −0.76 * . F 2.71 0.83 His 196 . . . . T T . 1.91−0.76 * * F 3.40 1.60 Gly 197 . . . . T T . 1.91 −0.50 * * F 3.06 2.54Lys 198 . . B . . . . 1.64 −0.93 . * F 2.12 3.71 Arg 199 . . B B . . .1.78 −0.67 . * F 1.58 2.02 Tyr 200 . . B B . . . 1.78 −0.74 . * . 1.433.16 Arg 201 . . B B . . . 1.81 −0.79 . * . 1.43 2.12 Val 202 . . B B .. . 2.16 −0.79 . * F 1.92 1.81 Pro 203 . . B . . T . 1.90 −0.39 . * F2.36 1.85 Ser 204 . . . . T T . 1.09 −0.71 . * F 3.40 1.46 Asp 205 . . .. . T C 0.48 0.07 . * F 1.96 1.71 Asn 206 . . . . . T C 0.07 0.07 . * F1.47 0.82 Pro 207 . . . . . . C 0.92 0.03 . . F 0.93 0.82 Phe 208 . . B. . . . 0.92 −0.36 . . F 0.99 0.85 Val 209 . . B . . . . 0.88 0.07 . . F0.33 0.82 Ser 210 . . B . . . . 0.29 0.10 . . F 0.61 0.52 Glu 211 . . B. . T . 0.26 0.17 . . F 1.09 0.61 Pro 212 . . . . T T . 0.26 −0.11 . . F2.52 1.12 Gly 213 . . . . T T . 0.37 −0.33 . . F 2.80 1.29 Ala 214 . . .. . T C 0.33 −0.21 . . . 2.02 0.75 His 215 . . . . . . C 0.39 0.47 . . .0.64 0.34 Pro 216 . . B B . . . −0.20 0.80 . . . −0.04 0.54 Ala 217 . .B B . . . −0.23 0.87 . . . −0.32 0.54 Ile 218 . . B B . . . −0.23 1.13 .. . −0.60 0.62 Tyr 219 . . B . . T . −0.53 1.06 . * . −0.20 0.40 Ala 220. . B . . T . −0.39 1.31 . . . −0.20 0.28 Tyr 221 . . B . . T . −0.180.81 * . . −0.20 0.77 Gly 222 . . B . . T . −0.19 0.53 * . . −0.20 0.79Ile 223 . . B B . . . 0.41 0.39 * * . −0.30 0.78 Arg 224 . . B B . . .0.77 0.80 * * . −0.60 0.52 Asn 225 . . . B T . . 0.69 0.04 * * . 0.251.03 Met 226 . . . B T . . 0.34 0.19 * * . 0.10 0.79 Trp 227 . . B B . .. −0.17 0.00 * * . 0.30 0.41 Arg 228 . . B . . . . 0.72 0.64 * * . −0.400.19 Cys 229 . . B . . . . 0.72 0.24 * * . 0.24 0.32 Ala 230 . . B . . .. 0.38 −0.37 * . . 1.18 0.59 Val 231 . . B . . . . 0.98 −0.86 * . . 1.820.30 Asp 232 . . . . T T . 1.06 −0.86 * . F 2.91 0.93 Arg 233 . . . . TT . 0.06 −1.00 * . F 3.40 1.42 Gly 234 . . . . T T . 0.41 −0.81 * * F3.06 1.34 Asp 235 . . . . . T C 1.11 −0.97 * . F 2.52 1.16 Pro 236 . . B. . . . 1.97 −0.97 * . F 1.78 1.16 Ile 237 . . B . . . . 1.62 −0.57 * *F 1.78 2.03 Thr 238 . . B . . . . 1.62 −0.57 * * F 1.78 1.20 Arg 239 . .B . . . . 1.62 −0.57 * * F 2.12 1.52 Gln 240 . . B . . . . 1.73−0.57 * * F 2.46 2.15 Gly 241 . . . . T T . 1.06 −1.26 . * F 3.40 2.92Arg 242 . . . . T T . 1.24 −1.06 . * F 3.06 1.05 Gly 243 . . . . T T .0.89 −0.27 . * F 2.27 0.52 Arg 244 . . B . . T . 0.43 −0.10 . * F 1.530.28 Ile 245 . . B . . . . 0.43 −0.10 . * . 0.84 0.14 Phe 246 . . B . .. . −0.08 −0.10 . * . 0.50 0.24 Cys 247 . . B . . T . −0.53 0.11 . * .0.40 0.09 Gly 248 . . B . . T . −0.19 0.54 . . . 0.40 0.13 Asp 249 . . .. T T . −0.30 0.26 . . F 1.55 0.26 Val 250 . . . . . T C 0.70 −0.13 . .F 2.25 0.77 Gly 251 . . . . . T C 0.70 −0.70 . * F 3.00 1.53 Gln 252 . .. . . T C 1.37 −0.34 * . F 2.25 0.80 Asn 253 . . . . . T C 1.71 −0.34 *. F 2.10 1.86 Arg 254 . . B . . T . 0.86 −0.99 * . F 1.90 3.25 Phe 255 AA . . . . . 1.71 −0.77 . . F 1.20 1.39 Glu 256 A A . . . . . 1.24 −1.17. * F 0.90 1.45 Glu 257 A A . . . . . 0.36 −0.89 . * . 0.60 0.61 Val 258A A . B . . . −0.46 −0.20 * * . 0.30 0.49 Asp 259 A A . B . . . −0.52−0.30 . * . 0.30 0.23 Leu 260 A A . B . . . −0.17 −0.30 * * . 0.30 0.27Ile 261 A A . B . . . −0.51 0.13 * * . −0.30 0.36 Leu 262 A . . . . T .−0.51 −0.09 * * . 0.70 0.21 Lys 263 . . . . T T . 0.10 0.31 * * F 0.650.42 Gly 264 . . . . T T . −0.24 0.39 * . F 0.65 0.93 Gly 265 . . . . TT . 0.28 0.13 * * F 0.80 1.12 Asn 266 . . . . . T C 1.28 0.36 * * F 0.450.59 Tyr 267 . . . . . T C 1.50 0.36 * * . 0.45 1.17 Gly 268 . . . . . TC 1.50 0.43 * * . 0.15 1.19 Trp 269 . . B . . T . 1.84 0.00 * * . 0.851.48 Arg 270 . A B . . . . 1.84 −0.40 * * . 0.45 1.63 Ala 271 . A B . .. . 1.14 −0.73 * * F 0.90 1.63 Lys 272 A A . . . . . 0.80 −0.37 * * F0.60 1.35 Glu 273 A A . . . . . 0.48 −0.79 * * F 0.75 0.69 Gly 274 A A .. . . . 0.52 −0.21 . * F 0.45 0.37 Phe 275 A A . . . . . 0.41 0.04 * * .−0.30 0.29 Ala 276 A A . . . . . 1.04 0.04 * . . −0.30 0.28 Cys 277 A .. . . T . 1.04 0.04 * . . 0.10 0.56 Tyr 278 A . . . . T . 0.23 −0.39 * .. 0.85 1.30 Asp 279 A . . . . T . −0.09 −0.49 * . F 1.00 1.06 Lys 280 A. . . . T . 0.58 −0.41 * . F 1.00 1.06 Lys 281 A A . . . . . 1.17 −0.49. . F 0.45 0.92 Leu 282 A A . . . . . 1.24 −0.84 . . . 0.60 0.89 Cys 283A A . . . . . 1.19 −0.34 . * . 0.30 0.45 His 284 . A B . . . . 0.38 0.04. * . −0.30 0.30 Asn 285 . . B . . T . 0.33 0.73 . * . −0.20 0.30 Ala286 A . . . . T . 0.29 0.04 . * . 0.10 0.94 Ser 287 A . . . . T . 0.24−0.53 . . . 1.15 1.15 Leu 288 A . . . . T . 0.10 −0.39 . . F 0.85 0.53Asp 289 . . B B . . . −0.08 −0.10 . . F 0.45 0.43 Asp 290 . . B B . . .−0.97 −0.17 . * F 0.45 0.50 Val 291 . . B B . . . −0.62 0.13 . . . −0.300.42 Leu 292 . . B B . . . −0.91 0.20 . . . −0.30 0.40 Pro 293 . . B B .. . −0.34 0.70 * . . −0.60 0.24 Ile 294 . . B B . . . −0.69 1.46 . . .−0.60 0.51 Tyr 295 . . B . . T . −0.72 1.24 . . . −0.20 0.61 Ala 296 . .B . . T . −0.46 1.06 . . . −0.20 0.54 Tyr 297 . . B . . T . −0.50 1.13. * . −0.20 0.77 Gly 298 . . B . . T . −0.63 1.09 * . . −0.20 0.37 His299 . . B B . . . 0.30 0.76 * . . −0.60 0.36 Ala 300 . . B B . . . 0.240.26 * . . −0.30 0.46 Val 301 . . B B . . . −0.02 −0.11 * . . 0.30 0.62Gly 302 . . B . . T . −0.09 0.10 * . F 0.25 0.34 Lys 303 . . B . . T .−0.09 0.09 * . F 0.25 0.48 Ser 304 . . B . . T . −0.40 0.01 * . F 0.250.64 Val 305 . . B . . T . −0.06 −0.20 . . F 0.85 0.64 Thr 306 . . B . .T . −0.06 0.13 . . F 0.25 0.50 Gly 307 . . B . . T . 0.04 0.77 . . F−0.05 0.28 Gly 308 . . B . . T . 0.11 1.14 . . F −0.05 0.59 Tyr 309 . .B . . T . 0.07 0.50 . . . −0.20 0.80 Val 310 . . B . . . . 0.26 0.44 * .. −0.40 0.80 Tyr 311 . . B . . T . 0.57 0.59 * . . −0.20 0.43 Arg 312 .. B . . T . 0.61 0.16 * . . 0.10 0.48 Gly 313 . . B . . T . 0.74 −0.21 *. F 1.13 0.87 Cys 314 . . B . . T . 0.99 −0.43 * * F 1.41 0.85 Glu 315 .. B . . . . 1.03 −0.79 * * F 1.79 0.70 Ser 316 . . . . . T C 1.28 −0.10. * F 2.17 0.58 Pro 317 . . . . T T . 0.82 −0.13 . * F 2.80 1.75 Asn 318. . . . T T . 0.36 −0.27 . . F 2.52 1.00 Leu 319 . . . . T T . 0.78 0.41. . F 1.19 0.62 Asn 320 . . . B T . . −0.11 0.79 . . . 0.36 0.63 Gly 321. . B B . . . −0.51 1.04 . . . −0.32 0.27 Leu 322 . . B B . . . −0.641.43 . . . −0.60 0.29 Tyr 323 . . B B . . . −0.64 1.17 . * . −0.60 0.18Ile 324 . . B B . . . −0.53 0.77 * . . −0.60 0.30 Phe 325 . . B B . . .−1.13 1.13 * . . −0.60 0.31 Gly 326 . . B B . . . −1.09 1.06 * . . −0.600.20 Asp 327 . . B . . . . −0.62 0.69 . * . −0.40 0.38 Phe 328 . . B . .. . −0.27 0.43 . * . −0.40 0.43 Met 329 A . . . . T . −0.19 −0.36 . * .0.70 0.85 Ser 330 . . . . . T C −0.09 −0.10 . * F 1.05 0.42 Gly 331 A .. . . T . −0.33 0.51 . . F −0.05 0.48 Arg 332 A . . . . T . −1.14 0.23 .. . 0.10 0.49 Leu 333 A A . . . . . −0.44 0.30 . . . −0.30 0.30 Met 334A A . . . . . 0.16 0.31 . * . −0.30 0.53 Ala 335 A A . . . . . 0.46−0.11 . * . 0.30 0.47 Leu 336 A A . . . . . 0.91 −0.11 . * . 0.30 0.95Gln 337 A A . . . . . 0.84 −0.80 . . . 0.75 1.87 Glu 338 A A . . . . .1.66 −1.41 . . F 0.90 3.71 Asp 339 A A . . . . . 2.30 −1.51 * . F 0.907.23 Arg 340 A A . . . . . 2.93 −2.20 * . F 0.90 8.35 Lys 341 A A . . .. . 3.46 −2.60 . * F 0.90 9.64 Asn 342 A . . . . T . 3.50 −1.69 * . F1.30 6.07 Lys 343 A . . . . T . 3.54 −1.69 * . F 1.30 6.20 Lys 344 A . .. . T . 3.54 −1.69 * . F 1.30 6.20 Trp 345 A . . . . T . 3.43 −1.29 * .F 1.30 6.68 Lys 346 A A . . . . . 2.58 −1.69 . . F 0.90 5.58 Lys 347 A A. . . . . 1.91 −1.00 . . F 0.90 2.30 Gln 348 . A B . . . . 1.06 −0.43 *. F 0.60 1.17 Asp 349 . A B . . . . 0.67 −0.66 . . F 0.75 0.48 Leu 350 .A B . . . . 0.66 −0.23 . . F 0.45 0.24 Cys 351 . A B . . . . 0.30 0.16 .. F −0.15 0.19 Leu 352 . A B . . . . −0.06 0.24 . . F −0.15 0.16 Gly 353. A . . T . . −0.36 0.73 . . F −0.05 0.28 Ser 354 . . . . T T . −1.020.43 . . F 0.35 0.70 Thr 355 . . . . T T . −0.80 0.43 . . F 0.35 0.45Thr 356 . . B . . T . −0.83 0.24 . . F 0.25 0.46 Ser 357 . . B . . T .−0.23 0.60 . . F −0.05 0.30 Cys 358 . . B . . . . −0.23 0.64 . . . −0.400.32 Ala 359 . . B . . . . −0.74 0.59 . . . −0.40 0.22 Phe 360 . . B . .T . −1.32 0.79 . . . −0.20 0.14 Pro 361 . . B . . T . −1.31 1.09 . . .−0.20 0.18 Gly 362 . . . . T T . −1.32 0.90 . . . 0.20 0.24 Leu 363 . .B . . T . −0.69 0.89 . . . −0.20 0.39 Ile 364 . . B . . . . −0.40 0.60 *. . −0.40 0.35 Ser 365 A . . . . T . 0.34 0.56 * * . −0.20 0.47 Thr 366A . . . . T . −0.14 0.13 * . F 0.40 1.13 His 367 A . . . . T . −0.690.23 * * F 0.40 1.40 Ser 368 . . B . . T . −0.77 0.23 * * F 0.25 0.73Lys 369 . . B B . . . −0.18 0.53 * * . −0.60 0.36 Phe 370 . . B B . . .−0.58 0.43 * * . −0.60 0.35 Ile 371 . . B B . . . −0.86 0.71 * * . −0.600.23 Ile 372 . . B B . . . −0.82 0.83 * * . −0.60 0.11 Ser 373 . A B . .. . −0.52 0.83 * . . −0.60 0.23 Phe 374 A A . . . . . −0.57 0.04 * * .−0.30 0.54 Ala 375 A A . . . . . −0.46 −0.64 . . . 0.75 1.35 Glu 376 A A. . . . . 0.09 −0.83 * . . 0.75 1.01 Asp 377 A A . . . . . 0.98 −0.79 *. F 0.90 1.16 Glu 378 A A . . . . . 0.47 −1.57 . . F 0.90 1.99 Ala 379 AA . . . . . 0.92 −1.39 . . F 0.75 0.95 Gly 380 A A . . . . . 0.81 −0.63. . F 0.75 0.89 Glu 381 A A . B . . . 0.00 0.16 . . . −0.30 0.44 Leu 382A A . B . . . −0.59 0.84 . . . −0.60 0.36 Tyr 383 A A . B . . . −0.900.84 . . . −0.60 0.37 Phe 384 A A . B . . . −0.61 0.90 . . . −0.60 0.31Leu 385 . A B B . . . −0.51 1.29 . . . −0.60 0.50 Ala 386 . A B B . . .−0.72 1.36 . . . −0.60 0.50 Thr 387 . A B B . . . −0.21 1.03 . . . −0.600.89 Ser 388 . A . . . . C −0.56 0.63 . . F −0.10 1.45 Tyr 389 . . . . .T C −0.10 0.44 . . F 0.30 1.45 Pro 390 . . . . T T . 0.12 0.70 . . F0.50 1.58 Ser 391 . . . . T T . 0.50 0.71 . . . 0.35 1.19 Ala 392 . . B. . T . 0.92 0.76 . . . 0.08 1.17 Tyr 393 . . B . . . . 0.88 0.00 . . .0.91 1.49 Ala 394 . . B . . T . 0.82 0.00 . * . 1.24 1.10 Pro 395 . . B. . T . 0.14 0.00 . * F 1.52 1.46 Arg 396 . . . . T T . 0.20 0.19 . . F1.30 0.65 Gly 397 . . B . . T . 0.83 0.19 . . F 0.92 1.01 Ser 398 . . BB . . . 0.38 −0.31 . . F 0.99 1.31 Ile 399 . . B B . . . 0.11 0.04 * . .−0.04 0.58 Tyr 400 . . B B . . . 0.32 0.69 * * . −0.47 0.43 Lys 401 . .B B . . . 0.00 0.26 * * . −0.30 0.54 Phe 402 . . B B . . . 0.04 0.30 * .. 0.19 1.19 Val 403 . . B B . . . 0.46 0.00 * . F 1.28 1.02 Asp 404 . .B . . T . 1.46 −0.76 * . F 2.17 1.00 Pro 405 . . B . . T . 1.11 −0.76 *. F 2.66 2.26 Ser 406 . . . . T T . 0.86 −1.04 * . F 3.40 3.07 Arg 407 .. . . T T . 1.34 −1.26 * . F 3.06 2.85 Arg 408 . . . . T . . 1.86 −0.83. . F 2.86 2.85 Ala 409 . . . . . . C 1.90 −0.83 . . F 2.66 2.10 Pro 410. . . . . T C 1.44 −1.21 * . F 2.86 2.15 Pro 411 . . . . T T . 1.79−0.64 * * F 2.91 0.59 Gly 412 . . . . T T . 1.43 −0.64 . * F 3.40 1.16Lys 413 . . . . T T . 1.37 −0.39 . * F 2.76 1.18 Cys 414 . . . . T T .1.74 −0.81 . * F 2.72 1.52 Lys 415 . . B . . T . 1.10 −0.81 . * F 1.982.38 Tyr 416 . . B . . T . 1.10 −0.60 . * F 1.49 0.88 Lys 417 . . B . .T . 0.59 −0.17 . * F 1.00 2.55 Pro 418 . . B B . . . 0.66 −0.10 . * F0.45 0.95 Val 419 . . B B . . . 1.01 −0.10 . * F 0.60 1.18 Pro 420 . . BB . . . 1.01 −0.37 . * F 0.79 0.85 Val 421 . . B B . . . 0.96 −0.37 . *F 1.28 1.10 Arg 422 . . B B . . . 0.96 −0.41 . * F 1.62 1.99 Thr 423 . .B . . T . 1.28 −1.06 * * F 2.66 2.58 Lys 424 . . . . T T . 1.24−1.49 * * F 3.40 6.80 Ser 425 . . . . T T . 1.24 −1.44 * * F 3.06 2.43Lys 426 . . . . T T . 1.40 −1.01 * * F 2.72 2.61 Arg 427 . . B . . . .1.40 −0.71 . * F 1.78 1.13 Ile 428 . . B . . . . 1.50 −0.71 * * F 1.441.65 Pro 429 . . B . . . . 0.64 −0.67 * * . 0.95 1.28 Phe 430 . . B . .. . 0.36 0.01 * * . −0.10 0.54 Arg 431 . A B . . . . 0.36 0.51 * * .−0.60 0.77 Pro 432 . A B . . . . −0.07 −0.17 * * F 0.60 1.00 Leu 433 A A. . . . . −0.03 −0.11 * * F 0.60 1.67 Ala 434 A A . . . . . −0.63−0.26 * * F 0.45 0.63 Lys 435 A A . . . . . 0.07 0.43 * * F −0.45 0.34Thr 436 A A . . . . . −0.86 0.00 * * . 0.30 0.68 Val 437 A A . . . . .−1.46 0.00 * . . 0.30 0.56 Leu 438 A A . . . . . −0.60 0.19 * . . −0.300.23 Asp 439 A A . . . . . −0.01 0.19 * . . −0.30 0.32 Leu 440 A A . . .. . −0.06 −0.30 * . . 0.30 0.74 Leu 441 A A . . . . . −0.04 −0.54 * . F0.90 1.56 Lys 442 A A . . . . . 0.81 −0.84 * . F 0.90 1.25 Glu 443 A A .. . . . 1.67 −0.84 * . F 0.90 2.63 Gln 444 A A . . . . . 1.08 −1.53 * .F 0.90 6.38 Ser 445 A A . . . . . 1.30 −1.71 * . F 0.90 3.22 Glu 446 A A. . . . . 2.22 −1.21 * . F 0.90 1.88 Lys 447 A A . . . . . 2.22 −1.21 *. F 0.90 2.13 Ala 448 A A . . . . . 1.92 −1.61 * . F 0.90 3.17 Ala 449 AA . . . . . 1.62 −1.61 * . F 0.90 2.46 Arg 450 A A . . . . . 1.62−1.23 * . F 0.90 1.65 Lys 451 A A . . . . . 1.03 −0.84 * . F 0.90 2.18Ser 452 A . . . . T . 0.68 −0.84 * . F 1.30 2.18 Ser 453 A . . . . T .0.46 −0.86 . . F 1.30 1.61 Ser 454 . . B . . T . 0.46 −0.17 . . F 0.850.66 Ala 455 . . B . . T . 0.04 0.33 . . F 0.25 0.50 Thr 456 . . B . . .. −0.34 0.33 . . . −0.10 0.50 Leu 457 . . B . . . . −0.26 0.37 . . .−0.10 0.37 Ala 458 . . B . . T . −0.54 0.41 . . F −0.05 0.57 Ser 459 . .B . . T . −0.24 0.41 . . F −0.05 0.40 Gly 460 . . . . . T C 0.00 0.33 .. F 0.45 0.83 Pro 461 . . . . . T C −0.50 0.07 * . F 0.45 0.81 Ala 462 .. . . . . C 0.01 0.26 * . F 0.25 0.50 Gln 463 A . . . . . . 0.60 0.26 .. F 0.05 0.68 Gly 464 . . B . . . . 0.94 −0.17 . . F 0.65 0.76 Leu 465 .. B . . . . 0.94 −0.60 . . F 1.10 1.50 Ser 466 A . . . . . . 0.86−0.67 * . F 0.95 0.86 Glu 467 A . . . . . . 1.14 −0.69 . * F 1.10 1.16Lys 468 A . . . . . . 1.19 −0.73 . . F 1.10 1.89 Gly 469 A . . . T T .1.58 −1.41 . . F 1.70 2.82 Ser 470 A . . . . T . 1.58 −1.80 . . F 1.303.26 Ser 471 A . . . . T . 1.29 −1.11 . . F 1.30 1.34 Lys 472 A . . . .T . 0.99 −0.61 . . F 1.30 1.37 Lys 473 . . B . . . . 0.73 −0.66 . . F1.10 1.37 Leu 474 . . B . . . . 0.77 −0.61 . . F 1.10 1.58 Ala 475 . . B. . . . 0.77 −0.51 * . F 1.40 1.14 Ser 476 . . B . . T . 0.77 −0.13 . .F 1.45 0.77 Pro 477 . . B . . T . 0.77 0.26 . . F 1.30 1.24 Thr 478 . .. . T T . 0.72 −0.43 . . F 2.60 2.46 Ser 479 . . . . . T C 1.22 −0.53 .. F 3.00 2.95 Ser 480 . . . . T T . 1.00 −0.43 * * F 2.60 2.76 Lys 481 .. B . . T . 1.41 −0.17 * * F 1.90 1.58 Asn 482 . . B . . T . 1.28−0.66 * * F 1.90 2.30 Thr 483 . . B . . T . 1.38 −0.61 * * F 1.94 1.70Leu 484 . . B . . . . 1.33 −0.57 * * F 1.78 1.32 Arg 485 . . B . . . .1.32 −0.14 * * F 1.67 0.81 Gly 486 . . B . . T . 1.32 −0.06 . * F 2.210.81 Pro 487 . . . . T T . 1.37 −0.54 . * F 3.40 1.96 Gly 488 . . . . TT . 1.72 −1.23 . * F 3.06 2.00 Thr 489 . . . . . T C 1.94 −1.23 . * F2.52 4.05 Lys 490 . A B . . . . 1.94 −1.16 . * F 1.58 2.65 Lys 491 . A B. . . . 1.43 −1.59 * * F 1.24 5.24 Lys 492 . A B . . . . 1.30 −1.37 * *F 0.90 2.69 Ala 493 . A B . . . . 1.43 −1.43 * * F 0.90 1.33 Arg 494 . AB . . . . 1.71 −1.00 * * F 0.90 1.03 Val 495 . A B . . . . 0.81−0.50 * * F 0.75 0.70 Gly 496 . . B . . T . 0.88 0.14 * * F 0.25 0.52Pro 497 . . B . . T . 0.83 −0.36 * * F 0.85 0.52 His 498 . . B . . T .1.08 0.04 * * F 0.74 1.20 Val 499 . . B . . T . 1.01 −0.17 * * F 1.681.20 Arg 500 . . B . . T . 1.98 −0.60 * * F 2.32 1.55 Gln 501 . . B . .T . 2.43 −1.03 * . F 2.66 2.24 Gly 502 . . . . T T . 2.69 −1.53 * . F3.40 5.90 Lys 503 A . . . . T . 2.42 −2.17 . . F 2.66 6.03 Arg 504 A . .. . . . 2.47 −1.79 . * F 2.12 4.66 Arg 505 . . B . . . . 2.40 −1.50 . .F 1.78 3.89 Lys 506 . . B . . . . 2.10 −1.93 . * F 1.44 3.89 Ser 507 . .B . . . . 2.41 −1.54 . . F 1.44 2.66 Leu 508 . . B . . . . 2.07 −1.04 .. F 1.78 1.85 Lys 509 . . B . . . . 1.61 −0.66 . . F 2.12 1.24 Ser 510 .. . . . . C 1.61 −0.23 * * F 2.21 0.91 His 511 . . . . T T . 0.97−0.61 * * F 3.40 2.17 Ser 512 . . . . . T C 1.38 −0.69 . * F 2.86 1.07Gly 513 . . . . T T . 1.98 −0.69 . * F 3.02 1.57 Arg 514 . . . . T T .1.63 −0.64 . * F 2.98 1.78 Met 515 . . . . . . C 1.34 −0.76 . * F 2.541.78 Arg 516 . . . . . T C 1.38 −0.64 . * F 2.70 1.82 Pro 517 . . . . .T C 1.68 −1.07 . * F 3.00 1.61 Ser 518 A . . . . T . 2.07 −0.67 . * F2.50 2.82 Ala 519 A . . . . T . 2.07 −1.29 . * F 2.20 2.88 Glu 520 A A .. . . . 2.08 −1.29 * * F 1.50 3.65 Gln 521 A A . . . . . 1.62 −1.21 * *F 1.51 2.75 Lys 522 A A . . . . . 1.94 −1.17 * . F 1.52 2.69 Arg 523 A A. . . . . 1.94 −1.67 * . F 1.83 3.04 Ala 524 . A . . T . . 1.72 −1.29 *. F 2.54 2.36 Gly 525 . . . . T T . 1.51 −1.00 * . F 3.10 0.97 Arg 526 .. . . T T . 1.12 −0.57 * . F 2.79 0.77 Ser 527 . . . . . T C 0.69−0.14 * . F 1.98 0.97 Leu 528 . . . . . T C 0.19 −0.21 * . . 1.67 1.25Pro 529 . . B . . . . 0.39 −0.21 * . . 0.81 0.82

TABLE II Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . .. . . C 0.69 −0.24 . * . 1.19 1.74 Arg 2 . . . . . . C 0.38 −0.24 . * .1.53 1.34 Pro 3 . . . . . T C 0.88 0.11 . . . 1.32 0.91 Pro 4 . . . . TT . 1.27 −0.31 . . . 2.61 1.80 Gly 5 . . . . T T . 0.96 −0.53 . . F 3.401.48 Phe 6 . . . . T T . 0.74 0.26 . . F 2.01 0.83 Arg 7 . A B . . . .−0.18 0.51 . . . 0.42 0.44 Asn 8 . A B . . . . −0.78 0.77 * . . 0.080.37 Phe 9 . A B . . . . −1.16 1.03 * . . −0.26 0.35 Leu 10 . A B . . .. −1.11 0.74 * . . −0.60 0.18 Leu 11 . A . . . . C −0.71 1.13 * . .−0.40 0.15 Leu 12 . A . . . . C −1.63 1.11 * . . −0.40 0.23 Ala 13 . A .. . . C −2.44 1.01 . . . −0.40 0.23 Ser 14 . A . . . . C −2.44 1.01 . .. −0.40 0.23 Ser 15 . A . . . . C −2.22 1.11 . . . −0.40 0.24 Leu 16 . AB . . . . −1.76 0.93 . . . −0.60 0.24 Leu 17 . A B . . . . −1.76 0.86 .. . −0.60 0.18 Phe 18 . A B . . . . −1.47 1.16 . . . −0.60 0.11 Ala 19 .A . . . . C −1.76 1.16 . . . −0.40 0.18 Gly 20 . A . . . . C −2.31 0.97. . . −0.40 0.22 Leu 21 . A . . . . C −1.71 0.93 * . . −0.40 0.19 Ser 22. A . . . . C −0.90 0.57 * . . −0.40 0.29 Ala 23 . A . . . . C −0.500.47 * . . −0.40 0.51 Val 24 . A . . . . C −0.61 0.43 * . . −0.40 0.83Pro 25 . . . . . T C −0.57 0.53 * . F 0.15 0.53 Gln 26 . . . . T T .0.03 0.53 * . F 0.35 0.71 Ser 27 . . . . T T . 0.03 0.46 * . F 0.50 1.48Phe 28 . . . . . T C −0.19 0.20 * * F 0.60 1.28 Ser 29 . . . . . T C0.78 0.46 * * F 0.15 0.61 Pro 30 . . . . . T C 0.69 0.06 * * F 0.45 0.89Ser 31 . . . . T T . 0.40 0.06 * * F 0.80 1.38 Leu 32 . . . . T T . 0.490.19 * * F 0.80 1.08 Arg 33 . . . . T . . 0.84 0.23 * * F 0.60 1.08 Ser34 . . . . T . . 0.56 0.23 * * F 0.45 0.80 Trp 35 . . . . . T C 0.180.34 * * F 0.45 0.98 Pro 36 . . . . . T C −0.19 0.16 * * F 0.45 0.50 Gly37 . . . . T T . 0.73 0.73 * * . 0.20 0.20 Ala 38 . . . . T T . −0.190.34 * * . 0.50 0.38 Ala 39 . . . . . . C −0.19 0.11 * . . 0.10 0.20 Cys40 . . B . T . . 0.21 0.07 * . . 0.30 0.27 Arg 41 . A B . . . . −0.17−0.36 * . . 0.30 0.53 Leu 42 . A . . . . C 0.18 −0.36 * . . 0.50 0.53Ser 43 . A . . . . C 0.47 −0.86 * * . 0.95 1.70 Arg 44 . A . . . . C1.06 −1.04 * . F 1.10 1.16 Ala 45 . A . . . . C 1.83 −1.04 . * F 1.102.44 Glu 46 . A . . T . . 1.83 −1.73 . * F 1.30 3.57 Ser 47 . A . . T .. 1.98 −2.11 . * F 1.30 3.57 Glu 48 . A . . T . . 2.39 −1.54 . * F 1.301.89 Arg 49 . A . . T . . 1.69 −2.04 . * F 1.30 2.14 Arg 50 . A . . T .. 2.07 −1.54 * * F 1.58 1.62 Cys 51 . A . . T . . 1.72 −1.50 * * . 1.711.44 Arg 52 . A . . T . . 2.02 −1.07 * * F 1.99 0.73 Ala 53 . . . . . TC 1.81 −0.67 * * F 2.47 0.64 Pro 54 . . . . T T . 1.49 −0.24 * * F 2.801.86 Gly 55 . . . . T T . 1.03 −0.39 . * F 2.52 1.47 Gln 56 . . . . . TC 1.11 0.04 * * F 1.44 1.44 Pro 57 . . . . . T C 0.41 0.04 * * F 1.010.94 Pro 58 . . . . T T . 0.19 0.11 . . F 0.93 0.96 Gly 59 . . . . T T .−0.27 0.37 . . F 0.65 0.46 Ala 60 . . B . . T . 0.04 0.54 . . . −0.200.16 Ala 61 . . B . . . . −0.30 0.61 . * . −0.40 0.14 Leu 62 . . B . . .. 0.02 0.61 . * . −0.40 0.14 Cys 63 . . . . T . . −0.11 0.19 . * . 0.610.27 His 64 . . . . T T . 0.34 0.11 . * . 1.12 0.26 Gly 65 . . . . T T .0.27 −0.39 . * F 2.18 0.63 Arg 66 . . . . T T . 0.86 −0.50 . * F 2.490.63 Gly 67 . . . . T T . 1.00 −1.07 . * F 3.10 0.77 Arg 68 . . . . T .. 1.32 −1.00 . * F 2.59 0.42 Cys 69 . . . . T T . 0.50 −1.00 . * . 2.330.21 Asp 70 . . . . T T . 0.18 −0.36 . * . 1.72 0.16 Cys 71 . . . . T T. −0.82 −0.21 . * . 1.41 0.04 Gly 72 . . . . T T . −1.14 0.47 . * . 0.200.06 Val 73 . . . B T . . −1.29 0.47 . * . −0.20 0.02 Cys 74 . . B B . .. −1.48 0.97 . . . −0.60 0.05 Ile 75 . . B B . . . −1.79 1.04 * . .−0.60 0.03 Cys 76 . . B B . . . −1.12 1.10 . . . −0.60 0.07 His 77 . . BB . . . −0.99 0.46 . . . −0.60 0.22 Val 78 . . . B T . . −0.48 0.31 . .. 0.10 0.48 Thr 79 . . . B . . C −0.41 0.06 . . F 0.05 0.89 Glu 80 . . .. . T C −0.22 0.10 * . F 0.45 0.64 Pro 81 . . . . T T . −0.26 0.39 . . F0.65 0.75 Gly 82 . . . . T T . −0.57 0.53 . . . 0.20 0.45 Met 83 . . . .T T . 0.08 0.47 * . . 0.20 0.26 Phe 84 . . . . T . . −0.42 0.90 . . .0.00 0.26 Phe 85 . . . . T . . −1.09 1.16 . . . 0.00 0.21 Gly 86 . . . .. T C −0.88 1.30 . . . 0.00 0.12 Pro 87 . . . . . T C −1.20 0.69 . . .0.00 0.23 Leu 88 . . . . T T . −0.63 0.47 * . . 0.20 0.14 Cys 89 . . . .T T . 0.07 0.19 * . . 0.50 0.20 Glu 90 . A . . T . . 0.48 −0.24 * . .0.70 0.22 Cys 91 . A . . T . . −0.03 0.24 . . . 0.10 0.28 His 92 . A . .T . . −0.49 0.20 * . . 0.10 0.39 Glu 93 . A . . T . . 0.32 0.20 * . .0.10 0.12 Trp 94 . A . . T . . 0.68 0.20 * . . 0.10 0.39 Val 95 . A . .T . . 0.43 0.11 * . . 0.10 0.42 Cys 96 . A . . T . . 1.10 0.37 * . .0.38 0.38 Glu 97 . A . . T . . 0.79 0.37 * . . 0.66 0.60 Thr 98 . A . .T . . 0.49 −0.11 * . F 1.69 0.80 Tyr 99 . . . . T T . 0.47 −0.37 . . F2.52 1.99 Asp 100 . . . . T T . 0.66 −0.46 . . F 2.80 1.66 Gly 101 . . .. T T . 0.73 0.11 . . F 1.77 0.62 Ser 102 . . . . T T . 0.39 0.13 . . F1.49 0.40 Thr 103 . . . . T . . 0.67 −0.20 . . F 1.61 0.24 Cys 104 . . .. T T . 0.57 0.30 . * . 0.78 0.32 Ala 105 . . . . T T . 0.61 0.30 . * .0.50 0.24 Gly 106 . . . . T T . 0.29 −0.09 . * . 1.10 0.33 His 107 . . .. T T . 0.59 0.00 . * . 0.50 0.33 Gly 108 . . . . T . . 0.23 −0.57 . * F1.35 0.55 Lys 109 . . . . T . . 0.56 −0.50 . * F 1.36 0.30 Cys 110 . . .. T T . 1.19 −0.50 . . . 1.72 0.22 Asp 111 . . . . T T . 0.87 −1.00 . *. 2.33 0.44 Cys 112 . . . . T T . 0.94 −0.86 . * . 2.64 0.12 Gly 113 . .. . T T . 0.62 −0.86 . * F 3.10 0.44 Lys 114 . . . . T . . 0.58 −0.86. * F 2.59 0.14 Cys 115 . . . . T . . 1.24 −0.86 . * F 2.28 0.44 Lys 116. . . . T . . 0.90 −1.03 . * F 1.97 0.76 Cys 117 . . . . T . . 1.28−1.03 . * F 1.94 0.38 Asp 118 . . . . T T . 1.38 −0.11 . * F 1.81 0.74Gln 119 . . . . T T . 0.99 0.07 . * F 1.49 0.58 Gly 120 . . . . T T .1.66 0.50 . * F 1.62 1.07 Trp 121 . . . . T T . 1.02 −0.07 . * F 2.801.07 Tyr 122 . . . . T . . 1.02 0.43 . . . 1.12 0.62 Gly 123 . . . . T .. 1.02 0.60 . . . 0.84 0.34 Asp 124 . . . . T . . 0.78 0.57 . . . 0.560.56 Ala 125 . . . . T . . 0.91 0.41 . . . 0.28 0.56 Cys 126 . . . . T .. 0.89 0.09 * . . 0.30 0.87 Gln 127 . . . . T . . 1.13 0.14 * . . 0.300.75 Tyr 128 . . . . . . C 0.81 0.54 . . . −0.05 1.20 Pro 129 . . . . TT . 0.81 0.61 . * F 0.50 1.20 Thr 130 . . . . T T . 0.59 0.04 . * F 0.801.15 Asn 131 . . . . T T . 0.94 0.33 . * F 0.65 0.61 Cys 132 . . . . T T. 0.99 0.06 . * . 0.50 0.57 Asp 133 . . . . T . . 1.28 −0.37 . . . 0.900.79 Leu 134 . . . . T . . 1.53 −0.86 . . F 1.69 0.98 Thr 135 . . . . T. . 1.54 −1.26 . . F 2.18 3.64 Lys 136 . . . . T . . 1.54 −1.44 . . F2.52 2.92 Lys 137 . . . . T . . 2.21 −1.04 * . F 2.86 5.70 Lys 138 . . .. T T . 1.61 −1.33 * . F 3.40 6.84 Ser 139 . . . . T T . 1.76 −1.20 * .F 3.06 3.39 Asn 140 . . . . T T . 2.11 −0.63 * . F 2.57 0.91 Gln 141 . .. . T T . 2.07 −0.63 * . F 2.57 0.91 Met 142 . . . . T . . 1.72 −0.23 *. . 2.07 1.09 Cys 143 . . . . T T . 1.68 −0.23 * . . 2.12 0.91 Lys 144 .. . . T T . 1.98 −0.23 * . F 2.61 0.91 Asn 145 . . . . T T . 1.09−0.63 * . F 3.40 1.53 Ser 146 . . . . T T . 0.20 −0.56 * . F 3.06 2.00Gln 147 . . . B T . . 0.13 −0.44 * . F 1.87 0.70 Asp 148 . . . B T . .0.50 0.13 * . F 0.93 0.23 Ile 149 . . B B . . . 0.46 0.11 * . . 0.040.23 Ile 150 . . B B . . . −0.13 0.13 . . . −0.30 0.22 Cys 151 . . B . .T . −0.18 0.23 . . . 0.10 0.13 Ser 152 . . . . T T . −0.49 0.66 . . .0.20 0.19 Asn 153 . . . . T T . −1.16 0.46 . . F 0.35 0.38 Ala 154 . . .. T T . −0.30 0.34 . . F 0.65 0.38 Gly 155 . . . . T . . −0.08 0.27 . .F 0.45 0.39 Thr 156 . . . . T . . 0.24 0.46 . . . 0.00 0.13 Cys 157 . .. . T T . 0.66 0.49 . . . 0.20 0.13 His 158 . . . . T T . −0.01 −0.01. * . 1.10 0.25 Cys 159 . . . . T T . 0.62 0.13 . * . 0.50 0.09 Gly 160. . . . T T . 0.30 −0.36 . * . 1.10 0.35 Arg 161 . . . . T . . 0.61−0.36 . * . 1.24 0.14 Cys 162 . . . . T T . 1.28 −0.86 . * F 2.23 0.43Lys 163 . . . . T T . 1.01 −1.03 . * F 2.57 0.69 Cys 164 . . . . T T .1.68 −1.07 . * F 2.91 0.47 Asp 165 . . . . T T . 1.68 −1.07 . * F 3.401.48 Asn 166 . . . . T T . 1.27 −1.21 . * F 2.91 0.73 Ser 167 . . . . TT . 1.59 −0.83 . . F 2.72 1.83 Asp 168 . . . . T T . 0.73 −0.97 . . F2.38 1.08 Gly 169 . . . . T T . 0.54 −0.29 . . F 1.59 0.56 Ser 170 . . .B T . . 0.30 −0.04 . . F 0.85 0.31 Gly 171 . . . B T . . −0.04 0.33 . *F 0.25 0.29 Leu 172 . . B B . . . 0.30 0.76 . * F −0.45 0.29 Val 173 . .B B . . . −0.40 0.33 . * . −0.30 0.43 Tyr 174 . . . B T . . −0.72 0.73. * . −0.20 0.38 Gly 175 . . . . T T . −0.42 0.87 . . . 0.20 0.24 Lys176 . . . . T T . −0.74 0.19 . . . 0.50 0.57 Phe 177 . . . . T T . 0.070.11 . . . 0.84 0.20 Cys 178 . . . . T T . 0.92 −0.64 * * . 2.08 0.33Glu 179 . . . . T . . 1.28 −1.07 * . . 2.22 0.28 Cys 180 . . . . T T .1.62 −1.07 * . . 2.76 0.62 Asp 181 . . . . T T . 0.91 −1.86 * * F 3.402.01 Asp 182 . . . . T T . 0.72 −1.86 * * F 2.91 0.62 Arg 183 . . . . TT . 1.39 −1.17 . * F 2.88 0.81 Glu 184 . . . . T . . 1.39 −1.74 . * .2.50 0.81 Cys 185 . . . . T . . 2.06 −1.74 . * . 2.47 0.81 Ile 186 . . .. T . . 1.74 −1.74 * * . 2.44 0.72 Asp 187 . . . . T T . 1.74 −1.26 . *F 3.10 0.60 Asp 188 . . . . . T C 1.63 −1.26 * * F 2.74 1.94 Glu 189 A .. . . T . 0.74 −1.83 * . F 2.23 4.79 Thr 190 A . . . . T . 0.74 −1.83 *. F 1.92 2.01 Glu 191 A . . . . . . 1.29 −1.26 * . F 1.26 0.65 Glu 192 A. . . . . . 0.94 −0.83 . . F 0.95 0.37 Ile 193 . . . . T . . 0.91 −0.40. . . 1.15 0.25 Cys 194 . . . . T T . 0.57 −0.39 . . . 1.60 0.20 Gly 195. . . . T T . 0.92 0.04 . . . 1.25 0.11 Gly 196 . . . . T T . 0.260.04 * . F 1.65 0.32 His 197 . . . . T T . 0.01 −0.07 . . F 2.50 0.32Gly 198 . . . . T T . 0.23 0.11 . . F 1.65 0.51 Lys 199 . . . . T T .0.56 0.26 . . . 1.25 0.28 Cys 200 . . . . T T . 0.90 0.26 . . . 1.000.20 Tyr 201 . . . . T T . 0.58 0.16 . . . 0.75 0.33 Cys 202 . . . . T T. 0.37 0.30 . . . 0.50 0.09 Gly 203 . . . . T T . 0.04 1.06 . * . 0.200.26 Asn 204 . . . . T T . 0.04 1.06 . * . 0.20 0.09 Cys 205 . . . . T T. 0.12 0.30 * * . 0.50 0.33 Tyr 206 . . . . T . . 0.02 0.23 . * . 0.300.34 Cys 207 . . . . T T . 0.40 0.23 * * . 0.50 0.21 Lys 208 . . . . T T. 0.71 0.74 * * . 0.20 0.40 Ala 209 . . . . T T . 0.37 0.67 * * . 0.200.35 Gly 210 . . . . T T . 1.03 0.34 * * . 0.81 0.65 Trp 211 . . . . T .. 1.32 −0.23 * * . 1.52 0.54 His 212 . . . . . T C 1.32 −0.23 * * . 1.981.07 Gly 213 . . . . T T . 1.28 −0.16 . * F 2.49 0.58 Asp 214 . . . . TT . 1.17 −0.59 . * F 3.10 0.96 Lys 215 . . . . T T . 1.51 −0.71 . * F2.79 0.61 Cys 216 . A . . T . . 1.13 −0.81 . * . 2.08 1.07 Glu 217 . A .. T . . 1.17 −0.67 . * . 1.62 0.34 Phe 218 . A . . T . . 0.62 −0.67 . *. 1.31 0.29 Gln 219 . A . . T . . 0.31 0.01 . * . 0.10 0.37 Cys 220 . A. . T . . 0.06 −0.07 . * . 0.70 0.31 Asp 221 . A . . T . . 0.43 0.36 . *. 0.10 0.56 Ile 222 . A . . . . C 0.43 0.49 . * . −0.06 0.34 Thr 223 . .. . . T C 0.83 0.09 . * F 1.28 1.09 Pro 224 . . . . T T . 0.88 −0.10 . .F 2.27 0.88 Trp 225 . . . . T T . 1.66 −0.10 . * F 2.76 2.50 Glu 226 . .. . T T . 1.77 −0.79 . * F 3.40 3.39 Ser 227 . . . . T T . 1.99 −1.27 .. F 3.06 4.29 Lys 228 . . . . T T . 1.99 −1.13 * * F 2.72 2.19 Arg 229 .. . . T T . 1.90 −1.56 * * F 2.38 1.82 Arg 230 . . . . T T . 1.98 −1.17. * F 2.38 1.82 Cys 231 . . . . T . . 1.98 −1.13 . . F 2.18 1.41 Thr 232. . . . T . . 1.93 −1.13 . * F 2.52 1.20 Ser 233 . . . . . T C 1.93−0.70 * * F 2.71 0.61 Pro 234 . . . . T T . 0.93 −0.70 * * F 3.40 2.27Asp 235 . . . . T T . 0.16 −0.59 * * F 3.06 1.10 Gly 236 . . . . T T .0.52 −0.50 . * F 2.58 0.44 Lys 237 . . . . T . . 0.83 −0.50 . * F 2.350.38 Ile 238 . . . . T . . 1.24 −0.53 . * . 2.47 0.37 Cys 239 . . . . TT . 1.11 −0.53 . * . 2.64 0.73 Ser 240 . . . . T T . 0.80 −0.53 . * F3.10 0.36 Asn 241 . . . . T T . 0.48 −0.04 . . F 2.49 0.74 Arg 242 . . .. T T . −0.42 −0.16 . . F 2.18 0.74 Gly 243 . . . B T . . −0.20 −0.09 .. F 1.47 0.41 Thr 244 . . . B T . . 0.12 0.10 * . F 0.56 0.14 Cys 245 .. . B T . . 0.42 0.13 * . . 0.10 0.07 Val 246 . . . B T . . −0.24 0.13 *. . 0.10 0.12 Cys 247 . . . . T T . −0.67 0.27 * * . 0.50 0.04 Gly 248 .. . . T T . −0.99 0.27 . . . 0.50 0.12 Glu 249 . . . . T T . −0.71 0.27. . . 0.50 0.09 Cys 250 . . . . T T . −0.04 0.13 . . . 0.50 0.22 Thr 251. . . . T . . −0.04 −0.44 . * . 0.90 0.37 Cys 252 . . . . T . . 0.62−0.23 . . . 0.90 0.16 His 253 . . . . T . . 0.76 −0.23 . . . 1.24 0.50Asp 254 . . . . T . . 0.44 −0.37 . * . 1.58 0.54 Val 255 . . . . T . .0.77 −0.37 . * . 2.07 1.44 Asp 256 . . . . . T C 1.08 −0.51 . * F 2.861.05 Pro 257 . . . . T T . 1.46 −1.01 * * F 3.40 1.05 Thr 258 . . . . TT . 1.14 −0.10 * * F 2.76 1.48 Gly 259 . . . . T T . 1.14 −0.31 . * F2.27 0.88 Asp 260 . . . . T . . 1.11 −0.31 . * F 1.73 0.95 Trp 261 . . .. T . . 1.08 −0.06 . * F 1.39 0.46 Gly 262 . . . . . . C 0.94 −0.04 . *F 0.85 0.63 Asp 263 . . . . T . . 1.26 −0.04 . * F 1.05 0.38 Ile 264 . .. . T . . 1.29 −0.04 . * . 0.90 0.60 His 265 . . . . T T . 0.62 −0.47 *. . 1.10 0.87 Gly 266 . . . . T T . 0.91 −0.33 * * F 1.25 0.28 Asp 267 .. . . T T . 0.59 −0.33 . * F 1.56 0.69 Thr 268 . . . . T T . 0.59 −0.44. * F 1.87 0.27 Cys 269 . . . . T T . 1.48 −0.94 * * . 2.33 0.46 Glu 270. . . . T T . 1.62 −1.37 . . . 2.64 0.48 Cys 271 . . . . T T . 1.97−1.37 * . F 3.10 0.65 Asp 272 . . . . T T . 1.30 −1.86 * . F 2.94 2.01Glu 273 . . . . T . . 1.72 −1.86 * * F 2.28 0.62 Arg 274 . . . . T T .1.80 −1.86 * . F 2.32 2.28 Asp 275 . . . . T T . 0.94 −1.93 * . F 2.011.38 Cys 276 . . . . T T . 1.37 −1.29 * . . 1.40 0.59 Arg 277 . . . . TT . 1.37 −0.53 * . . 1.40 0.47 Ala 278 . . B B . . . 1.48 −0.53 * . .0.60 0.47 Val 279 . . B B . . . 1.12 −0.53 * . . 1.09 1.73 Tyr 280 . . .B T . . 0.82 −0.34 * * . 1.53 1.38 Asp 281 . . . . T T . 1.49 0.04 * . .1.67 1.83 Arg 282 . . . . T T . 1.38 −0.46 * * F 2.76 4.12 Tyr 283 . . .. T T . 1.27 −1.10 * * F 3.40 4.39 Ser 284 . . . . T T . 1.46 −1.07 * *F 3.06 2.28 Asp 285 . . . . T . . 1.40 −0.50 * . F 2.07 0.62 Asp 286 . .. . T . . 1.06 −0.11 * . F 1.73 0.53 Phe 287 . . . . T . . 0.91−0.44 * * . 1.24 0.39 Cys 288 . . . . T T . 0.81 −0.33 . . . 1.10 0.32Ser 289 . . . . T T . 1.11 0.10 . . . 0.50 0.19 Gly 290 . . . . T T .0.44 0.50 . * F 0.35 0.38 His 291 . . . . T T . 0.44 0.29 . * F 0.870.38 Gly 292 . . . . T . . 0.48 0.11 . * F 0.89 0.46 Gln 293 . . . . T .. 0.80 0.30 * * . 0.96 0.25 Cys 294 . . . . T T . 1.21 0.30 * * . 1.380.18 Asn 295 . . . . T T . 0.89 −0.20 . * . 2.20 0.36 Cys 296 . . . . TT . 0.92 −0.06 . * . 1.98 0.11 Gly 297 . . . . T T . 0.60 −0.46 * * .2.04 0.34 Arg 298 . . . . T . . 0.64 −0.46 * * . 1.90 0.11 Cys 299 . . .. T T . 0.72 −0.86 * * . 2.46 0.43 Asp 300 . . . . T T . 0.38 −0.93 * *. 2.52 0.44 Cys 301 . . . . T T . 0.76 −0.93 * * . 2.80 0.22 Lys 302 . .. . T T . 0.86 −0.01 * * . 2.22 0.43 Ala 303 . . . . T . . 0.40 0.17 * *. 1.14 0.40 Gly 304 . . . . T . . 1.11 0.60 . * . 0.56 0.75 Trp 305 . .. . T . . 1.16 0.03 . * . 0.92 0.75 Tyr 306 . . . . T . . 1.16 0.03 . *. 1.13 1.48 Gly 307 . . . . T T . 1.11 0.10 * . F 1.67 0.80 Lys 308 . .. . T T . 1.67 −0.33 * . F 2.76 1.32 Lys 309 . . . . T T . 1.80 −0.74 .. F 3.40 1.15 Cys 310 . . . . T T . 2.09 −1.07 . . F 3.06 1.79 Glu 311 .. . . T . . 2.03 −1.10 . . F 2.52 1.55 His 312 . . . . . T C 1.71 −0.71. . F 2.18 1.04 Pro 313 . . . . T T . 1.36 −0.14 . . F 1.74 1.04 Gln 314. . . . T T . 0.50 −0.23 . . F 1.25 0.87 Ser 315 . . . . T T . 0.87 0.46. . F 0.35 0.52 Cys 316 . . . B T . . 0.28 0.34 . . F 0.25 0.45 Thr 317. . . B . . C 0.31 0.41 . . . −0.40 0.27 Leu 318 . . . B . . C 0.52 0.01. . . −0.10 0.34 Ser 319 . . . B . . C 0.22 −0.37 . * . 0.65 1.11 Ala320 A A . . . . . −0.37 −0.56 * * F 0.90 1.03 Glu 321 A A . . . . . 0.41−0.36 * * F 0.45 0.87 Glu 322 A A . . . . . 0.77 −1.04 * * F 0.90 1.28Ser 323 . A . . T . . 0.91 −1.43 * * F 1.64 2.53 Ile 324 . A . . T . .1.21 −1.36 * * F 1.83 0.78 Arg 325 . A . . T . . 1.46 −0.96 * . F 2.170.78 Lys 326 . A . . T . . 1.16 −0.53 * . F 2.51 0.58 Cys 327 . . . . TT . 0.86 −0.53 * . F 3.40 1.11 Gln 328 . . . . T T . 1.16 −0.83 * * F2.91 0.76 Gly 329 . . . . T T . 1.23 −0.83 * * F 2.57 0.63 Ser 330 . . .. T T . 0.91 −0.14 * * F 1.93 0.97 Ser 331 . . . . T . . 0.20 −0.29 . *F 1.39 0.87 Asp 332 . . . . T . . 0.57 −0.11 * * F 1.05 0.47 Leu 333 . .. . . . C 0.22 −0.16 * * F 1.16 0.47 Pro 334 . . . . T . . 0.68−0.11 * * F 1.67 0.35 Cys 335 . . . . T T . 0.63 −0.50 . * F 2.18 0.41Ser 336 . . . . T T . 0.98 −0.07 . * F 2.49 0.49 Gly 337 . . . . T T .0.31 −0.76 . * F 3.10 0.63 Arg 338 . . . . T T . 1.12 −0.61 . * F 2.790.63 Gly 339 . . . . T . . 0.67 −1.19 . * F 2.56 0.82 Lys 340 . . . . T. . 0.99 −1.00 * * F 2.53 0.44 Cys 341 . . . . T T . 1.33 −1.00 * * F2.70 0.22 Glu 342 . . . . T T . 1.01 −1.00 * * . 2.52 0.45 Cys 343 . . .. T T . 0.59 −0.86 * * . 2.80 0.12 Gly 344 . . . . T T . 0.27 −0.37 . .. 2.22 0.33 Lys 345 . . . . T . . −0.02 −0.37 . . . 1.74 0.10 Cys 346 .. . . T . . 0.43 0.39 . . . 0.86 0.29 Thr 347 . . . . T . . 0.22 0.24 .. . 0.58 0.46 Cys 348 . . . . T . . 0.54 0.24 . * . 0.64 0.36 Tyr 349 .. B . . . . 0.89 0.67 . * . 0.28 0.66 Pro 350 . . . . . T C 0.96 0.10 .. F 1.47 0.76 Pro 351 . . . . T T . 1.73 −0.39 * . F 2.76 2.78 Gly 352 .. . . T T . 1.19 −0.96 * . F 3.40 3.47 Asp 353 . . . . T T . 1.61−1.07 * . F 3.06 1.67 Arg 354 . . . . T . . 1.51 −0.74 * * F 2.52 1.69Arg 355 . . . . T . . 1.77 −0.74 * * F 2.18 1.69 Val 356 . . . . T . .1.67 −1.17 * * . 1.69 2.02 Tyr 357 . . . . T . . 1.34 −0.69 * * . 1.351.49 Gly 358 . . . . T T . 1.34 −0.11 * * F 1.25 0.41 Lys 359 . . . . TT . 0.57 −0.11 * . F 1.25 0.95 Thr 360 . . . . T T . 0.46 −0.19 * * F1.59 0.33 Cys 361 . . . . T T . 1.31 −0.94 * * F 2.23 0.55 Glu 362 . . .. T . . 1.67 −1.37 * . . 2.22 0.46 Cys 363 . . . . T T . 2.12 −1.37 * .. 2.76 0.62 Asp 364 . . . . T T . 1.41 −1.86 * . F 3.40 2.28 Asp 365 . .. . T T . 1.72 −1.86 * . F 2.91 0.70 Arg 366 . . . . T T . 2.39 −1.86 *. F 3.03 2.28 Arg 367 . . . . T . . 1.58 −2.43 * . F 2.80 2.28 Cys 368 .. . . T . . 2.24 −1.74 . . F 2.77 1.12 Glu 369 . . . . T . . 1.90 −1.74. . F 2.59 0.96 Asp 370 . . . . T T . 1.04 −1.31 * . F 3.10 0.48 Leu 371. . . . T T . 0.08 −0.67 * . F 2.79 0.67 Asp 372 . . . . T T . −0.70−0.60 . . F 2.48 0.29 Gly 373 . . . . T T . −0.38 −0.03 * . . 1.72 0.09Val 374 . . B . . . . −0.72 0.40 * . . 0.21 0.11 Val 375 . . B . . . .−0.76 0.14 . * . −0.10 0.07 Cys 376 . . . . T T . −0.29 0.64 . . . 0.200.09 Gly 377 . . . . T T . −0.60 0.64 . . . 0.20 0.12 Gly 378 . . . . TT . −0.92 0.49 . . F 0.35 0.23 His 379 . . . . T T . −0.37 0.41 . . F0.35 0.23 Gly 380 . . . . T . . −0.18 0.23 . . F 0.45 0.32 Thr 381 . . .. T . . 0.14 0.37 * . F 0.45 0.17 Cys 382 . . . . T T . 0.60 0.37 * . .0.50 0.12 Ser 383 . . . . T T . 0.28 −0.13 * . . 1.28 0.25 Cys 384 . . .. T T . −0.54 0.01 * . . 0.86 0.09 Gly 385 . . . . T T . −0.87 0.17 * .. 1.04 0.13 Arg 386 . . . . T . . −0.56 0.17 * . . 1.02 0.05 Cys 387 . .B . T . . 0.22 −0.21 * . . 1.80 0.16 Val 388 . . B . . . . 0.18 −0.79 *. . 1.52 0.32 Cys 389 . . B . . . . 0.56 −0.79 * * . 1.34 0.16 Glu 390 .. . . T T . 0.20 0.13 * . . 0.86 0.32 Arg 391 . . . . T T . −0.26 0.34 .. . 0.68 0.38 Gly 392 . . . . T T . 0.46 0.13 . . . 0.50 0.69 Trp 393 .. . . T T . 0.50 −0.44 . . . 1.10 0.80 Phe 394 . . . . T . . 0.50 0.24 *. . 0.30 0.34 Gly 395 . . . . T . . 0.50 0.81 * . . 0.00 0.18 Lys 396 .. . . T . . 0.36 0.79 * . . 0.00 0.30 Leu 397 . . . . T . . 0.490.37 * * . 0.64 0.47 Cys 398 . . . . T . . 0.89 0.01 * * . 0.98 0.74 Gln399 . . . . T . . 1.63 −0.41 * . . 1.92 0.72 His 400 . . . . . T C 1.31−0.41 * * . 2.41 1.75 Pro 401 . . . . T T . 1.27 −0.53 * * F 3.40 1.75Arg 402 . . . . T T . 1.48 −0.70 * . F 3.06 1.63 Lys 403 . . . . T T .1.83 −0.49 . . F 2.42 1.18 Cys 404 . . . . T . . 1.83 −0.50 * . . 2.031.11 Asn 405 . A . . . . C 1.87 −0.93 . . . 1.14 0.98 Met 406 . A . . .. C 2.08 −0.93 * . F 0.95 0.85 Thr 407 . A . . . . C 1.67 −0.53 * . F1.44 2.73 Glu 408 A A . . . . . 1.67 −0.71 . * F 1.58 2.28 Glu 409 A A .. . . . 2.33 −1.11 * . F 1.92 4.61 Gln 410 . A . . T . . 1.52 −1.33 * .F 2.66 5.13 Ser 411 . . . . T T . 1.46 −1.13 * . F 3.40 2.44 Lys 412 . .. . T T . 1.77 −0.56 * . F 2.91 0.76 Asn 413 . . . . . T C 1.47 −0.56 *. F 2.62 0.76 Leu 414 . . . . . T C 0.88 −0.57 * . . 2.38 0.76 Cys 415 .. . . T . . 0.88 −0.46 * . . 1.99 0.38 Glu 416 . . . . T . . 0.83−0.46 * . F 2.05 0.40 Ser 417 . . . . T T . −0.10 −0.43 * . F 2.50 0.48Ala 418 . . . . T T . −0.91 −0.43 * . F 2.25 0.62 Asp 419 . . . . T T .−0.77 −0.31 * . F 2.00 0.30 Gly 420 . . . . T T . −0.40 0.26 * . . 1.000.12 Ile 421 . . B . . . . −0.74 0.26 * . . 0.15 0.16 Leu 422 . . B . .. . −0.40 0.19 * * . 0.15 0.09 Cys 423 . . . . T T . −0.16 0.19 * * .1.00 0.19 Ser 424 . . . . T T . −0.46 0.19 * * F 1.40 0.27 Gly 425 . . .. T T . −0.78 −0.11 . * F 2.25 0.43 Lys 426 . . . . T T . 0.08 −0.23 . *F 2.50 0.43 Gly 427 . . . . T . . 0.22 −0.30 . * F 2.05 0.44 Ser 428 . .. . T . . 0.54 −0.11 * * F 1.80 0.24 Cys 429 . . . . T . . 0.89−0.11 * * . 1.40 0.12 His 430 . . . . T T . 0.57 −0.11 * . . 1.35 0.24Cys 431 . . . . T T . −0.37 0.03 * . . 0.50 0.10 Gly 432 . . . . T T .−0.69 0.33 * . . 0.50 0.12 Lys 433 . . . . T T . −0.69 0.33 * . . 0.500.05 Cys 434 . . . . T . . −0.61 0.21 * . . 0.30 0.12 Ile 435 . A . . T. . −0.58 0.14 * . . 0.10 0.13 Cys 436 . A B . . . . 0.09 −0.29 * . .0.30 0.11 Ser 437 . A . . . . C 0.14 −0.29 * . . 0.50 0.35 Ala 438 . A .. . . C −0.14 0.06 * . . −0.10 0.52 Glu 439 . A . . T . . −0.37 0.13 . .. 0.25 1.53 Glu 440 . A . B T . . 0.22 0.24 . . . 0.10 0.80 Trp 441 . A. B T . . 0.54 0.24 . * . 0.25 1.06 Tyr 442 . A . B T . . 0.84 0.17 . *. 0.10 0.61 Ile 443 . . . B T . . 0.73 0.17 . * . 0.10 0.61 Ser 444 . .. B T . . 0.07 0.96 . * . −0.20 0.50 Gly 445 . . . . T . . 0.07 0.61 . *F 0.15 0.17 Glu 446 . . . . T . . −0.31 −0.14 . * . 0.90 0.41 Phe 447 .. . . T . . −0.07 −0.26 * . . 1.24 0.16 Cys 448 . . . . T T . 0.82−0.64 * * . 2.08 0.28 Asp 449 . . . . T T . 1.23 −1.07 * . . 2.42 0.27Cys 450 . . . . T T . 1.58 −1.07 * . . 2.76 0.60 Asp 451 . . . . T T .0.91 −1.86 * . F 3.40 1.87 Asp 452 . . . . T T . 1.61 −1.86 * . F 2.910.60 Arg 453 . . . . T T . 2.32 −1.86 * * F 2.72 1.87 Asp 454 . . . . TT . 2.29 −2.43 * * F 2.72 2.24 Cys 455 . . . . T T . 2.96 −1.93 . . F2.72 1.83 Asp 456 . . . . T . . 2.61 −1.93 . . F 2.52 1.56 Lys 457 . . .. T . . 1.80 −1.50 * . F 2.71 0.92 His 458 . . . . T T . 0.80 −0.81 * .F 3.40 1.42 Asp 459 . . . . T T . 0.13 −0.70 . . F 2.91 0.60 Gly 460 . .. . T T . 0.49 −0.13 . . . 2.12 0.16 Leu 461 . . B . . T . 0.14 0.36 . .. 0.78 0.17 Ile 462 . . B . . . . 0.10 0.29 . . . 0.24 0.10 Cys 463 . .. . T T . −0.21 0.69 * . . 0.20 0.16 Thr 464 . . . . T T . −1.10 0.69 *. F 0.35 0.20 Gly 465 . . . . T T . −1.42 0.69 * . F 0.35 0.20 Asn 466 .. . . T T . −0.91 0.57 * . F 0.35 0.20 Gly 467 . . . . T . . −0.69 0.39. . F 0.45 0.18 Ile 468 . . . . T . . −0.37 0.47 * . . 0.00 0.10 Cys 469. . . . T T . −0.06 0.47 * . . 0.42 0.06 Ser 470 . . . . T T . −0.380.47 * . . 0.64 0.10 Cys 471 . . . . T T . −0.38 0.61 . . . 0.86 0.08Gly 472 . . . . T T . −0.70 −0.07 . . . 1.98 0.24 Asn 473 . . . . T T .−0.10 −0.07 . . . 2.20 0.10 Cys 474 . . . . T T . 0.57 0.46 . . . 1.080.19 Glu 475 . . . . T T . 0.52 −0.11 . . . 1.76 0.32 Cys 476 . . . . TT . 0.90 −0.11 . . . 1.54 0.20 Trp 477 . . . . T T . 1.24 0.40 . . .0.42 0.39 Asp 478 . . . . T T . 0.90 0.23 . . . 0.50 0.36 Gly 479 . . .. T T . 1.57 0.66 . . F 0.35 0.67 Trp 480 . . . . T T . 0.98 0.49 . . F0.50 1.02 Asn 481 . . . . . T C 0.98 0.07 . . F 0.45 0.62 Gly 482 . . .. . T C 1.27 0.64 * . F 0.15 0.33 Asn 483 . . . . . T C 0.38 0.21 * . .0.30 0.55 Ala 484 . . . . . T C 0.43 −0.01 . . . 0.90 0.24 Cys 485 . A .. T . . −0.09 0.50 . . . −0.20 0.25 Glu 486 . A B . . . . −0.43 0.76 . .. −0.60 0.13 Ile 487 . A . . T . . −0.39 0.79 . . . −0.20 0.13 Trp 488 .A . . T . . −0.39 0.67 . . . −0.20 0.32 Leu 489 . A . . . . C −0.04 0.10. . . −0.10 0.32 Gly 490 . . . . T T . 0.41 0.86 . * F 0.56 0.72 Ser 491. . . . . T C 0.02 0.60 . . F 0.72 1.05 Glu 492 . . . . . T C 0.52 0.11. . F 1.23 1.63 Tyr 493 . . . . . T C 0.42 −0.14 . . . 1.89 2.11 Pro 494. . . . T . . 0.84 −0.14 . . . 2.10 2.01

TABLE III Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A A .. . . . 0.10 −0.19 . . . 0.30 0.92 Glu 2 A A . . . . . −0.32 −0.11 * * .0.30 0.72 Thr 3 A A . . . . . 0.18 0.14 * . . −0.30 0.47 Gly 4 A A . . .. . 0.68 −0.29 * . . 0.30 0.93 Ala 5 A A . . . . . 0.86 −0.90 * . F 0.901.05 Leu 6 A A . . . . . 1.46 −0.47 . . F 0.60 1.12 Arg 7 . A B . . . .0.64 −0.56 . . F 0.90 1.96 Arg 8 . . B . . . . 0.14 −0.30 * . F 0.801.60 Pro 9 . A B . . . . 0.28 −0.11 . . F 0.60 1.60 Gln 10 . A B . . . .0.06 −0.37 . . F 0.60 1.26 Leu 11 . A B . . . . 0.06 0.31 * . . −0.300.53 Leu 12 . A B . . . . −0.87 1.00 . . . −0.60 0.28 Pro 13 . A B . . .. −1.79 1.26 . * . −0.60 0.14 Leu 14 . A B . . . . −2.39 1.54 . . .−0.60 0.14 Leu 15 . A B . . . . −3.06 1.54 . . . −0.60 0.14 Leu 16 . A B. . . . −2.59 1.43 . . . −0.60 0.05 Leu 17 . A B . . . . −2.12 1.43 . .. −0.60 0.06 Leu 18 . A B . . . . −2.58 1.17 . . . −0.60 0.07 Cys 19 . .B . . T . −1.98 1.06 * * . −0.20 0.04 Gly 20 . . . . T T . −1.060.80 * * . 0.20 0.08 Gly 21 . . . . T T . −0.83 0.11 . * F 0.65 0.20 Cys22 . . B . . T . −0.37 −0.07 . * F 0.85 0.37 Pro 23 . . B . . . . 0.10−0.21 * * F 0.96 0.37 Arg 24 . . . . T T . 0.10 −0.21 . * F 1.87 0.37Ala 25 . . . . T T . 0.44 −0.07 . * F 2.18 0.37 Gly 26 . . . . T T .0.79 −0.24 . * F 2.49 0.38 Gly 27 . . . . T T . 1.14 −0.67 . * F 3.100.34 Cys 28 . . . . T . . 1.01 −0.19 . * F 2.29 0.48 Asn 29 . . . . . TC 0.30 −0.26 . * F 1.98 0.48 Glu 30 . . B . . T . 0.08 −0.07 . . F 1.470.48 Thr 31 . . B . . T . 0.42 0.19 * . F 0.56 0.74 Gly 32 . . B . . T .0.88 −0.39 * . F 0.85 0.80 Met 33 A A . . . . . 0.73 −0.79 * . . 0.600.91 Leu 34 A A . . . . . 0.52 −0.10 * * . 0.30 0.52 Glu 35 A A . . . .. −0.29 −0.16 . * . 0.30 0.81 Arg 36 A A . . . . . −0.64 0.10 * * .−0.30 0.67 Leu 37 A A . . . . . −0.64 0.06 * * . −0.30 0.44 Pro 38 A A .. . . . 0.00 −0.20 * * . 0.30 0.25 Leu 39 A A . . . . . 0.22 −0.20 * * .0.30 0.26 Cys 40 A A . . . . . −0.48 0.30 * * . −0.30 0.31 Gly 41 A A .. . . . −1.18 0.40 * * . −0.30 0.18 Lys 42 A A . . . . . −0.37 0.47 * *. −0.60 0.21 Ala 43 A A . . . . . −0.76 −0.21 * * . 0.30 0.67 Phe 44 A A. . . . . −0.54 −0.17 * * . 0.30 0.67 Ala 45 A A . . . . . −0.220.01 * * . −0.30 0.33 Asp 46 A A . . . . . 0.17 0.44 * * . −0.60 0.32Met 47 A A . . . . . −0.73 −0.06 * * . 0.30 0.75 Met 48 A A . . . . .−0.14 −0.20 * * . 0.30 0.55 Gly 49 A A . . . . . −0.30 −0.70 * * . 0.600.55 Lys 50 A . . B . . . 0.00 −0.06 . * . 0.30 0.41 Val 51 A . . B . .. 0.04 0.24 . * . −0.30 0.44 Asp 52 A . . B . . . 0.36 −0.37 . * . 0.300.89 Val 53 A . . B . . . 0.29 0.11 . * . −0.30 0.47 Trp 54 A . . B . .. 0.63 0.69 . * . −0.60 0.34 Lys 55 A . . B . . . −0.22 0.44 . * . −0.600.32 Trp 56 A . . B . . . 0.33 1.13 . . . −0.60 0.36 Cys 57 A . . B . .. 0.33 0.87 . . . −0.60 0.46 Asn 58 . . . B . . C 0.49 −0.04 * . . 0.500.40 Leu 59 . . . B . . C −0.11 0.74 * . . −0.40 0.33 Ser 60 . . . B . .C −1.01 0.51 * . . −0.40 0.43 Glu 61 . . B B . . . −0.97 0.59 * . .−0.60 0.20 Phe 62 . . B B . . . −0.54 0.94 . . . −0.60 0.38 Ile 63 . . BB . . . −0.54 1.01 * . . −0.60 0.44 Val 64 . . B B . . . −0.03 0.63 * .. −0.60 0.44 Tyr 65 . . B B . . . −0.43 1.01 . . . −0.60 0.68 Tyr 66 . .B B . . . −0.74 1.01 * . . −0.60 0.84 Glu 67 . . . B T . . −0.04 0.81 *. . −0.05 1.63 Ser 68 . . . B T . . 0.18 0.57 . . . −0.05 1.68 Phe 69 .. . . T T . 0.72 0.39 * . . 0.50 0.57 Thr 70 . . . . T T . 0.97 0.11 * .F 0.65 0.48 Asn 71 . . . . . T C 0.61 0.11 . . F 0.45 0.62 Cys 72 A . .. . T . 0.61 0.34 . * F 0.25 0.71 Thr 73 A A . . . . . 0.32 −0.44 . * F0.45 0.85 Glu 74 A A . . . . . 1.02 −0.43 * * . 0.30 0.53 Met 75 A A . .. . . 0.48 −0.43 . * . 0.45 1.60 Glu 76 A A . . . . . −0.38 −0.36 . * .0.30 0.82 Ala 77 A A . . . . . −0.06 −0.20 . * . 0.30 0.35 Asn 78 A A .. . . . −0.41 0.23 . * . −0.30 0.35 Val 79 . A B . . . . −0.66 0.19 . *. −0.30 0.11 Val 80 . A B . . . . −0.34 0.94 . * . −0.60 0.17 Gly 81 . AB . . . . −0.56 1.36 . * . −0.60 0.11 Cys 82 . . . . T . . 0.03 1.39 * .. 0.00 0.23 Tyr 83 . . . . T . . −0.18 1.14 * . . 0.00 0.50 Trp 84 . . B. . T . −0.13 0.93 . . . −0.20 0.78 Pro 85 . . . . . T C 0.13 1.19 * . F0.30 1.20 Asn 86 . . . . . T C 0.48 1.11 * . F 0.15 0.77 Pro 87 . . . .. T C 0.80 0.76 * . F 0.30 1.27 Leu 88 . . . . . . C 0.34 0.27 * . F0.25 0.81 Ala 89 . . . . . . C −0.26 0.63 * . F −0.05 0.44 Gln 90 . . BB . . . −0.36 0.91 * . . −0.60 0.20 Gly 91 . . B B . . . −0.70 0.97 * .. −0.60 0.35 Phe 92 . . B B . . . −1.38 0.71 * . . −0.60 0.34 Ile 93 . .B B . . . −0.60 0.90 * . . −0.60 0.14 Thr 94 . . B B . . . 0.10 1.00 * .. −0.60 0.19 Gly 95 . . B B . . . 0.10 0.57 * . . −0.60 0.43 Ile 96 . .B B . . . −0.26 0.19 * . . −0.15 1.06 His 97 . . B B . . . −0.26 0.29 *. . −0.30 0.64 Arg 98 . . . B T . . 0.33 0.59 * . . −0.20 0.56 Gln 99 .. . B T . . 0.64 0.54 * . . −0.05 1.06 Phe 100 . . . B T . . 0.32 0.26 *. . 0.25 1.26 Phe 101 . . . . T T . 0.90 0.33 * . . 0.50 0.34 Ser 102 .. . . T T . 0.08 0.81 . * . 0.20 0.29 Asn 103 . . . . T T . −0.03 1.06 .. . 0.20 0.25 Cys 104 . . . . T T . 0.08 0.27 . * . 0.50 0.47 Thr 105 .. . . T . . −0.08 −0.51 . . . 1.20 0.69 Val 106 . A . . T . . 0.59 −0.26. * . 0.70 0.32 Asp 107 . A B . . . . 0.08 −0.16 . * . 0.30 0.81 Arg 108. A B . . . . 0.08 −0.04 . * . 0.30 0.46 Val 109 . A B . . . . 0.74−0.53 . * . 0.75 1.08 His 110 . A B . . . . 0.84 −1.17 . * . 0.75 1.08Leu 111 . A . . . . C 1.49 −0.74 . * . 0.80 0.85 Glu 112 . A . . . . C1.49 −0.31 . * F 0.80 1.78 Asp 113 . A . . . . C 1.38 −0.96 * * F 1.102.18 Pro 114 . . . . . T C 1.38 −1.46 * * F 1.50 4.58 Pro 115 . . . . TT . 0.60 −1.50 * . F 1.70 1.96 Asp 116 A . . . . T . 0.52 −0.81 * . F1.15 0.97 Glu 117 A . . . . T . 0.31 −0.13 * * . 0.70 0.44 Val 118 A . .B . . . −0.50 −0.13 * . . 0.30 0.44 Leu 119 . . B B . . . −1.18 0.13 . .. −0.30 0.22 Ile 120 . . B B . . . −1.82 0.81 . . . −0.60 0.09 Pro 121 .. B B . . . −2.71 1.46 . . . −0.60 0.09 Leu 122 . . B B . . . −2.92 1.50. * . −0.60 0.07 Ile 123 . . B B . . . −2.92 1.24 . . . −0.60 0.16 Val124 . . B B . . . −2.97 1.20 . . . −0.60 0.08 Ile 125 . . B B . . .−2.89 1.41 . . . −0.60 0.07 Pro 126 . . B B . . . −2.99 1.41 . * . −0.600.08 Val 127 . . B B . . . −3.03 1.21 . . . −0.60 0.16 Val 128 . . B B .. . −2.73 1.21 . * . −0.60 0.17 Leu 129 . . B B . . . −2.48 1.03 . . .−0.60 0.11 Thr 130 . . B B . . . −2.18 1.21 . * . −0.60 0.15 Val 131 . .B B . . . −2.31 1.07 . . . −0.60 0.20 Ala 132 A . . B . . . −2.27 0.86 .. . −0.60 0.25 Met 133 A . . B . . . −2.27 0.86 . . . −0.60 0.14 Ala 134A . . B . . . −2.31 1.01 . . . −0.60 0.14 Gly 135 A . . B . . . −2.291.01 * * . −0.60 0.10 Leu 136 A . . B . . . −1.32 1.43 * * . −0.60 0.11Val 137 A . . B . . . −1.03 0.81 . * . −0.60 0.21 Val 138 A . . B . . .−0.39 0.70 * . . −0.26 0.29 Trp 139 . . B B . . . 0.31 0.27 * . . 0.380.70 Arg 140 . . B B . . . 0.34 −0.41 . . F 1.62 1.84 Ser 141 . . B . .T . 1.16 −0.57 . . F 2.66 3.58 Lys 142 . . . . T T . 1.70 −1.21 . * F3.40 5.68 Arg 143 . . . . T T . 1.74 −1.64 * . F 3.06 4.19 Thr 144 . . .. T T . 1.22 −0.96 . * F 2.72 2.58 Asp 145 . A . . T . . 0.72 −0.66 . .F 1.98 1.06 Thr 146 . A B . . . . 0.63 −0.23 * . F 0.79 0.69 Leu 147 . AB . . . . 0.20 0.20 * . . −0.30 0.61 Leu 148 . A B . . . . −0.30 0.14. * . −0.30 0.47

TABLE IV Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A A .. . . . −0.76 0.27 . * . −0.30 0.49 Arg 2 A A . . . . . −1.07 0.34 * * .−0.30 0.39 Leu 3 A A . . . . . −1.49 0.70 * * . −0.60 0.26 Leu 4 A A . .. . . −1.40 0.96 . * . −0.60 0.22 Ala 5 A A . . . . . −1.82 0.73 . * .−0.60 0.15 Phe 6 A A . . . . . −2.03 1.41 * * . −0.60 0.15 Leu 7 A A . .. . . −2.73 1.41 . * . −0.60 0.15 Ser 8 A A . . . . . −2.73 1.23 . . .−0.60 0.15 Leu 9 A A . . . . . −2.78 1.41 . . . −0.60 0.14 Leu 10 A A .. . . . −3.00 1.27 * . . −0.60 0.13 Ala 11 A A . . . . . −2.30 1.27 * .. −0.60 0.08 Leu 12 A A . . . . . −1.49 1.29 . . . −0.60 0.17 Val 13 A A. . . . . −1.50 0.60 . . . −0.60 0.35 Leu 14 A A . . . . . −1.03 0.40 .. . −0.02 0.50 Gln 15 A A B . . . . −0.53 0.33 . . F 0.41 0.60 Glu 16 A. . . . T . −0.53 0.13 . . F 1.24 1.17 Thr 17 A . . . . T . −0.02 −0.01. * F 2.12 1.43 Gly 18 . . . . T T . 0.02 −0.31 . . F 2.80 1.11 Thr 19 A. . . . T . 0.62 −0.03 * * F 1.97 0.53 Ala 20 A . . . . . . 0.73 0.40 .. F 0.89 0.57 Ser 21 . . . . . . C 0.78 −0.09 . . F 1.56 1.12 Leu 22 . A. . . . C 1.09 −0.51 * . F 1.38 1.55 Pro 23 A A . . . . . 1.54 −1.00 * .F 0.90 2.66 Arg 24 A A . . . . . 1.90 −1.50 * * F 0.90 3.88 Lys 25 A A .. . . . 2.60 −1.89 . . F 0.90 9.42 Glu 26 A A . . . . . 3.01 −2.57 . . F0.90 11.93 Arg 27 A A . . . . . 3.82 −3.00 . . F 0.90 11.93 Lys 28 A A .. . . . 4.03 −3.00 . . F 0.90 10.33 Arg 29 A A . . . . . 3.92 −3.00 . .F 0.90 10.33 Arg 30 A A . . . . . 3.28 −2.60 . * F 0.90 9.13 Glu 31 A A. . . . . 3.07 −1.99 * . F 0.90 4.52 Glu 32 A A . . . . . 3.07 −1.56 . .F 1.24 3.57 Gln 33 A A . . . . . 3.02 −1.56 * . F 1.58 3.57 Met 34 . A .. . . C 2.57 −1.56 * . F 2.12 3.57 Pro 35 A . . . . T . 2.46 −1.13 * . F2.66 2.04 Arg 36 . . . . T T . 2.16 −1.13 * . F 3.40 1.97 Glu 37 A . . .. T . 1.46 −1.14 * . F 2.66 2.66 Gly 38 . . . . T T . 1.46 −0.97 * * F2.72 1.49 Asp 39 A . . . . . . 1.20 −1.40 * . F 1.78 1.32 Ser 40 A . . .. . . 0.60 −0.76 * . F 1.29 0.57 Phe 41 . . B . . . . 0.28 −0.07 . . .0.50 0.47 Glu 42 . . B . . . . −0.53 −0.07 . . . 0.50 0.44 Val 43 . . B. . . . −0.08 0.61 . . . −0.40 0.27 Leu 44 . . B . . . . −0.08 0.23 . *. −0.10 0.61 Pro 45 A . . . . . . 0.22 −0.16 . * . 0.50 0.56 Leu 46 A .. . . T . 0.07 −0.16 . * . 0.85 1.27 Arg 47 A . . . . T . −0.74 −0.16. * F 1.00 1.14 Asn 48 . . B . T T . 0.11 −0.16 . * F 1.25 0.61 Asp 49 .. B . . T . 0.71 −0.19 . * F 1.00 1.19 Val 50 . . B . . . . 0.92 −0.44. * F 0.65 0.94 Leu 51 . . B . . . . 1.73 −0.44 . * F 0.95 0.97 Asn 52 .. B . . T . 1.38 −0.44 . . F 1.45 0.94 Pro 53 . . . . . T C 1.03 0.31 .. F 1.50 1.98 Asp 54 . . . . T T . 1.03 0.10 . * F 2.00 2.37 Asn 55 . .. . . T C 1.03 −0.59 . . F 3.00 2.56 Tyr 56 . . B B . . . 0.96 −0.34 . *F 1.80 1.23 Gly 57 . . B B . . . 0.96 −0.09 . . . 1.20 0.52 Glu 58 . . BB . . . 0.36 −0.09 * . . 0.90 0.53 Val 59 . . B B . . . 0.06 0.20 * . .0.00 0.28 Ile 60 . . B B . . . 0.06 −0.17 . . . 0.30 0.38 Asp 61 . . B B. . . 0.06 −0.20 . . . 0.30 0.35 Leu 62 . . B . . T . 0.40 0.56 . . .−0.20 0.75 Ser 63 . . . . . T C 0.40 −0.09 . . . 1.05 1.85 Asn 64 . . .. . T C 0.44 −0.77 . . F 1.50 1.91 Tyr 65 A . . . . T . 1.02 −0.09 . * F1.34 1.91 Glu 66 A . . . . . . 1.02 −0.29 . . F 1.48 2.06 Glu 67 A . . .. . . 1.59 −0.67 * . F 2.12 2.14 Leu 68 . . B . . . . 1.54 −0.31 * . F2.16 2.14 Thr 69 . . . . T T . 1.54 −0.64 * . F 3.40 1.22 Asp 70 . . . .T T . 1.79 −0.64 * . F 3.06 1.18 Tyr 71 . . . . T T . 0.98 −0.24 * . F2.42 2.48 Gly 72 . . . . T T . 0.77 −0.24 * . F 2.08 1.42 Asp 73 A . . .. . . 1.58 −0.30 * . F 1.23 1.31 Gln 74 A . . . . . . 1.03 −0.30 * * F0.98 1.45 Leu 75 . . B . . . . 1.08 −0.41 . * F 1.07 1.09 Pro 76 . . B .. . . 0.47 −0.84 . * F 1.46 1.30 Glu 77 . . B B . . . 0.50 −0.20 . * F0.90 0.56 Val 78 . . B B . . . 0.20 −0.11 . * F 0.81 0.98 Lys 79 . . B B. . . −0.61 −0.41 . * F 0.72 0.85 Val 80 . . B B . . . −0.39 −0.16 . * F0.63 0.40 Thr 81 . . B B . . . −0.39 0.34 . * F −0.06 0.55 Ser 82 . . B. . . . −0.98 0.13 . * F 0.05 0.42 Leu 83 . . B . . . . −0.43 0.63 . * .−0.40 0.58 Ala 84 . . B . . . . −0.78 0.47 . * . −0.40 0.58 Pro 85 A . .. . . . −0.81 0.37 . . . −0.10 0.58 Ala 86 . . B B . . . −0.80 0.67 . .F −0.45 0.49 Thr 87 . . B B . . . −0.71 0.37 . . F −0.15 0.65 Ser 88 . .B B . . . −0.49 0.30 . . F 0.13 0.65 Ile 89 . . B B . . . 0.14 0.37 . .F 0.41 0.65 Ser 90 . . B . . T . 0.06 −0.13 . . F 1.69 0.90 Pro 91 . . .. . T C 0.33 −0.23 . . F 2.17 0.90 Ala 92 . . . . T T . 0.33 −0.13 . . F2.80 1.86 Lys 93 . . B . . T . 0.04 −0.33 . . F 2.12 2.00 Ser 94 . . B .. . . 0.72 −0.21 . . F 1.64 1.31 Thr 95 . . B . . . . 0.68 −0.21 . . F1.36 2.00 Thr 96 . . B . . . . 0.58 −0.29 . . F 0.93 0.99 Ala 97 . . B .. . . 0.96 0.20 . . F 0.20 1.07 Pro 98 . . B . . . . 0.61 0.24 . . F0.48 1.14 Gly 99 . . . . T . . 0.61 0.14 . . F 1.16 1.06 Thr 100 . . . .. T C 0.92 0.04 . . F 1.44 1.41 Pro 101 . . . . . T C 1.02 −0.06 . . F2.32 1.46 Ser 102 . . . . T T . 1.30 −0.06 . . F 2.80 2.28 Ser 103 . . .. . T C 0.91 0.00 . . F 2.32 2.28 Asn 104 . . . . . T C 0.94 0.13 . . F1.44 1.46 Pro 105 . . . . . T C 1.37 0.19 . . F 1.36 1.57 Thr 106 . . .. T T . 1.37 −0.20 . . F 2.08 2.30 Met 107 . . B . . T . 1.36 −0.16 . .F 1.60 2.21 Thr 108 . . B . . . . 1.34 −0.07 . . F 1.60 2.07 Arg 109 . .B . . T . 0.76 −0.01 . . F 2.00 2.07 Pro 110 . . B . . T . 0.62 0.00 * .F 1.80 2.11 Thr 111 . . B . . T . 0.12 −0.19 . . F 1.60 1.45 Thr 112 . .B . . T . −0.09 0.01 . . F 0.65 0.61 Ala 113 . A B . . . . −0.59 0.70 .. F −0.25 0.32 Gly 114 . A B . . . . −1.00 0.96 . . . −0.60 0.19 Leu 115. A B . . . . −1.09 0.86 . . . −0.60 0.17 Leu 116 . A B . . . . −0.780.76 . . . −0.60 0.23 Leu 117 . A B . . . . −0.68 0.66 . * F −0.45 0.40Ser 118 . A B . . . . −0.09 0.66 . . F −0.29 0.75 Ser 119 . . B . . . .0.22 0.37 . . F 0.52 1.46 Gln 120 . . B . . T . 0.69 0.19 . . F 0.882.41 Pro 121 . . . . T T . 0.69 −0.07 . . F 2.04 1.78 Asn 122 . . . . TT . 1.29 0.23 . * F 1.60 1.10 His 123 . . . . T T . 1.28 0.27 . . F 1.290.98 Gly 124 . . . . T . . 0.91 0.36 . . . 0.78 0.91 Leu 125 . . . . . TC 0.10 0.50 . . . 0.32 0.30 Pro 126 . . . . T T . −0.54 0.79 . . . 0.360.18 Thr 127 . . . . T T . −1.21 0.93 . . . 0.20 0.14 Cys 128 . . B . .T . −2.03 1.07 . . . −0.20 0.09 Leu 129 . . B B . . . −2.36 1.03 . . .−0.60 0.04 Val 130 . . B B . . . −2.36 1.17 . . . −0.60 0.02 Cys 131 . .B B . . . −2.49 1.37 . . . −0.60 0.02 Val 132 . . B B . . . −2.48 1.23 .. . −0.60 0.03 Cys 133 . . B B . . . −2.11 0.93 . . . −0.60 0.05 Leu 134. . B B . . . −2.16 0.67 . . . −0.60 0.13 Gly 135 . . . . T T . −1.540.74 . * F 0.35 0.13 Ser 136 . . . . T T . −1.54 0.86 . * F 0.35 0.39Ser 137 . . B . . T . −0.69 0.86 . . F −0.05 0.25 Val 138 . . B . . T .−0.02 0.17 . . . 0.10 0.43 Tyr 139 . . B . . . . −0.10 −0.26 . . . 0.500.53 Cys 140 . . B . . T . 0.24 0.04 . * . 0.10 0.28 Asp 141 . . B . . T. −0.27 −0.34 . * . 0.70 0.63 Asp 142 . . B . . T . 0.03 −0.30 . * F0.85 0.33 Ile 143 . . B . . T . 0.89 −1.06 . . F 1.30 1.07 Asp 144 . A B. . . . 0.24 −1.63 . . F 0.90 1.07 Leu 145 . A B . . . . 0.70 −0.94 . .F 0.75 0.45 Glu 146 . A B . . . . 0.49 −0.51 . * F 0.75 0.99 Asp 147 . AB . . . . −0.32 −0.77 . * F 0.99 0.92 Ile 148 . A B . . . . 0.36−0.09 * * F 0.93 0.92 Pro 149 . . . . . . C 0.47 −0.34 * . F 1.57 0.82Pro 150 . . . . . . C 1.39 −0.34 * . F 1.81 0.96 Leu 151 . . . . . T C1.08 −0.34 * . F 2.40 2.68 Pro 152 . . B . . T . 0.49 −0.54 * . F 2.262.50 Arg 153 . . . . T T . 1.13 −0.47 * . F 2.12 1.63 Arg 154 . . B . .T . 0.53 −0.14 * . F 1.48 3.11 Thr 155 . . B B . . . 0.50 −0.14 * . .0.69 1.66 Ala 156 . . B B . . . 0.72 0.19 * * . −0.15 1.33 Tyr 157 . . BB . . . 1.04 0.69 * * . −0.60 0.68 Leu 158 . . B B . . . 0.23 0.69 * * .−0.60 0.93 Tyr 159 . . B B . . . 0.12 0.99 * * . −0.60 0.80 Ala 160 . .B B . . . 0.54 0.89 * * . −0.60 0.82 Arg 161 . . B B . . . 0.24 0.13 * *. −0.15 1.94 Phe 162 . . B B . . . 0.19 0.13 * * . −0.30 0.87 Asn 163 .. B B . . . 1.11 −0.24 * * . 0.45 1.15 Arg 164 . . B B . . . 0.47 −0.74. * F 1.08 1.15 Ile 165 . . B B . . . 1.17 −0.06 . * F 0.81 0.93 Ser 166. . . B . . C 0.47 −0.84 * * F 1.64 1.13 Arg 167 . . B B . . . 1.17−0.74 * * F 1.47 0.59 Ile 168 . . B B . . . 1.17 −0.74 * * F 1.80 1.45Arg 169 . . B B . . . 0.36 −1.43 * . . 1.47 1.80 Ala 170 A A . . . . .1.29 −1.03 * * F 1.29 0.80 Glu 171 A A . . . . . 1.24 −1.03 . * F 1.262.27 Asp 172 A A . . . . . 0.32 −1.29 . * F 1.08 1.15 Phe 173 A A . . .. . 0.90 −0.60 . * F 0.75 0.94 Lys 174 A A . . . . . 0.83 −0.61 . * F0.75 0.78 Gly 175 A A . . . . . 0.61 −0.61 * * F 0.75 0.94 Leu 176 A A .. . . . 0.66 0.07 * * F −0.15 0.89 Thr 177 A A . . . . . 0.77 −0.71 * *F 0.75 0.89 Lys 178 A A . . . . . 0.58 −0.71 * * F 0.90 1.76 Leu 179 A A. . . . . 0.53 −0.46 * * F 0.60 1.50 Lys 180 . A B . . . . 0.07−1.14 * * F 0.90 1.73 Arg 181 . A B . . . . 0.58 −0.94 * * F 0.75 0.71Ile 182 . A B . . . . 0.89 −0.56 . * F 0.90 1.16 Asp 183 . A B . . . .0.84 −0.84 . * . 0.60 0.93 Leu 184 . . B . . T . 0.84 −0.44 * * F 0.850.77 Ser 185 . . B . . T . −0.09 0.24 * * F 0.25 0.90 Asn 186 . . . . .T C −0.50 0.24 . * F 0.45 0.38 Asn 187 . . . . . T C 0.09 0.63 * * F0.15 0.62 Leu 188 . . B . . . . −0.80 0.33 * * . −0.10 0.62 Ile 189 . .B . . . . 0.01 0.63 * . . −0.40 0.27 Ser 190 . . B . . . . 0.31 0.23 * .F 0.05 0.28 Ser 191 . . B . . . . 0.31 0.23 * * F 0.05 0.54 Ile 192 . .B . . . . −0.28 −0.46 * . F 0.80 1.29 Asp 193 . . B . . T . −0.17−0.64 * . F 1.15 0.98 Asn 194 A . . . . T . 0.83 −0.24 * * F 0.85 0.63Asp 195 A . . . . T . 0.32 −0.63 * . F 1.30 1.76 Ala 196 A . . . . T .−0.19 −0.63 * . . 1.00 0.87 Phe 197 A A . . . . . 0.67 0.06 * . . −0.300.45 Arg 198 A A . . . . . 0.08 0.16 * . . −0.30 0.36 Leu 199 A A . . .. . −0.73 0.66 * * . −0.60 0.36 Leu 200 A A . . . . . −0.73 0.84 * . .−0.60 0.35 His 201 A A . . . . . −0.14 0.46 * * . −0.60 0.31 Ala 202 A A. . . . . −0.26 0.46 * * . −0.60 0.62 Leu 203 A A . . . . . −1.260.46 * * . −0.60 0.62 Gln 204 A A . . . . . −1.26 0.46 * . . −0.60 0.32Asp 205 . A B . . . . −0.66 0.64 . . . −0.60 0.26 Leu 206 . A B . . . .−0.62 0.57 . . . −0.60 0.49 Ile 207 . A B . . . . −0.03 −0.11 . . . 0.300.49 Leu 208 . . B . . T . 0.78 −0.11 * . F 0.85 0.47 Pro 209 A . . . .T . −0.03 0.29 . . F 0.25 0.99 Glu 210 A . . . . T . −0.03 0.29 . * F0.40 1.16 Asn 211 A . . . . T . 0.19 −0.40 . . F 1.00 2.44 Gln 212 A A .. . . . 0.27 −0.59 . * F 0.90 1.60 Leu 213 A A . . . . . 0.87 −0.33 . .. 0.30 0.76 Glu 214 A A . . . . . 0.22 0.10 . . . −0.30 0.73 Ala 215 . AB . . . . −0.59 0.34 . . . −0.30 0.31 Leu 216 . A B . . . . −0.80 0.63 .. . −0.60 0.31 Pro 217 . . B . . . . −1.10 0.37 . . . −0.10 0.28 Val 218. . B . . . . −0.63 0.76 * . . −0.40 0.37 Leu 219 . . B . . T . −1.520.69 * . F −0.05 0.44 Pro 220 . . . . . T C −0.93 0.69 * * F 0.15 0.20Ser 221 . . . . . T C −0.82 0.26 * . F 0.45 0.47 Gly 222 . . B . . T .−1.42 0.40 * . F 0.25 0.49 Ile 223 . A B . . . . −0.57 0.40 * . F −0.150.26 Glu 224 . A B . . . . −0.61 −0.03 . * . 0.30 0.33 Phe 225 . A B . .. . −0.29 0.23 . * . −0.30 0.25 Leu 226 . A B . . . . −0.80 −0.20 . * .0.30 0.69 Asp 227 A A . . . . . −0.46 −0.20 * * . 0.30 0.33 Val 228 A A. . . . . 0.54 0.20 * . . −0.30 0.61 Arg 229 A A . . . . . −0.27 −0.59. * . 0.75 1.45 Leu 230 A A . . . . . 0.43 −0.59 . * . 0.88 0.71 Asn 231A A . . . . . 0.94 −0.19 * * . 1.01 1.67 Arg 232 . A . . T . . 0.64−0.44 * * F 1.84 1.14 Leu 233 . A . . T . . 1.16 −0.06 * . F 2.12 1.85Gln 234 . . . . T T . 0.16 −0.31 * . F 2.80 1.14 Ser 235 . . . . T T .0.97 −0.03 * . F 2.37 0.41 Ser 236 . . . . . T C 0.76 0.37 * * F 1.290.86 Gly 237 . . . . T T . 0.06 0.11 * * F 1.21 0.76 Ile 238 . A B . . .. 0.28 0.21 . . F 0.13 0.58 Gln 239 . A B . . . . −0.42 0.33 . . F −0.150.43 Pro 240 . A B . . . . −0.01 0.73 * * F −0.45 0.38 Ala 241 A A . . .. . −0.30 0.30 * * . −0.15 1.06 Ala 242 A A . . . . . −0.56 0.11 * . .−0.30 0.62 Phe 243 A A . . . . . 0.33 0.33 * * . −0.30 0.40 Arg 244 A A. . . . . 0.38 −0.10 * * . 0.30 0.68 Ala 245 A A . . . . . −0.22−0.60 * * . 0.75 1.35 Met 246 A A . . . . . 0.37 −0.41 * * . 0.45 1.28Glu 247 A A . . . . . 0.26 −0.80 * * . 0.75 1.13 Lys 248 A A . . . . .0.14 −0.01 * * . 0.30 0.97 Leu 249 A A . . . . . −0.21 0.17 * * . −0.300.81 Gln 250 A A . . . . . −0.43 0.31 . . . −0.30 0.73 Phe 251 A A . . .. . −0.13 1.00 . . . −0.60 0.30 Leu 252 . A B . . . . −0.13 1.39 . * .−0.60 0.49 Tyr 253 . . B . . . . −0.18 0.70 . * . −0.40 0.47 Leu 254 . .B . . . . −0.18 0.70 * . . −0.23 0.88 Ser 255 . . B . . T . −0.99 0.60 *. . 0.14 0.88 Asp 256 . . . . T T . −0.29 0.60 * . . 0.71 0.46 Asn 257 .. . . T T . 0.22 −0.16 * . F 1.93 0.94 Leu 258 . . B . . T . −0.42−0.46 * . F 1.70 0.94 Leu 259 . . B . . . . 0.18 −0.16 * . F 1.33 0.39Asp 260 . . B . . . . 0.13 0.27 * . F 0.56 0.38 Ser 261 . . B . . . .−0.08 0.30 * . F 0.39 0.45 Ile 262 . . B . . T . −0.89 0.04 * . F 0.420.85 Pro 263 . . B . . T . −0.29 0.04 * . F 0.25 0.42 Gly 264 . . . . .T C 0.31 0.47 * . F 0.15 0.48 Pro 265 . . . . . T C 0.01 0.51 . * F 0.301.07 Leu 266 . . . . . . C −0.50 0.21 * * F 0.42 0.93 Pro 267 . . . . .T C 0.50 0.47 * * F 0.49 0.77 Pro 268 . . . . T T . 0.41 0.04 * * F 1.160.98 Ser 269 . . B . . T . −0.10 0.00 * * F 1.68 1.59 Leu 270 . . B . .T . 0.08 −0.04 * * F 1.70 0.76 Arg 271 . . B . . . . 0.08 0.03 * * F0.73 0.67 Ser 272 . . B . . . . 0.29 0.29 . . . 0.41 0.41 Val 273 . . B. . . . 0.50 0.30 . * . 0.24 0.87 His 274 . . B . . . . 0.80 0.01 . * .0.07 0.71 Leu 275 . . B . . T . 0.80 0.41 * . . −0.20 0.86 Gln 276 . . .. . T C −0.20 0.71 * . F 0.15 0.95 Asn 277 . . . . . T C 0.10 0.76 . * F0.15 0.49 Asn 278 . . . . . T C 0.64 0.26 * * F 0.60 1.03 Leu 279 A A .. . . . 0.08 0.06 * . . −0.30 0.86 Ile 280 A A . . . . . 0.89 0.27 * * .−0.30 0.53 Glu 281 . A B . . . . 1.00 0.27 * . . −0.30 0.57 Thr 282 A A. . . . . 1.00 −0.13 * . . 0.45 1.35 Met 283 . A B . . . . 0.14 −0.81 *. F 0.90 3.21 Gln 284 . A B . . . . 0.26 −0.86 * . F 0.90 1.38 Arg 285 .A B . . . . 0.48 −0.07 * . F 0.45 0.83 Asp 286 . A . . T . . 0.48 0.01 *. . 0.10 0.45 Val 287 . A B . . . . 0.58 −0.60 * . . 0.60 0.43 Phe 288 AA . . . . . 1.18 −0.57 * . . 0.60 0.34 Cys 289 A A . . . . . 1.18−0.57 * . . 0.60 0.35 Asp 290 A A . . . . . 1.03 −0.57 * . F 0.75 0.82Pro 291 A A . . . . . 1.08 −0.71 . . F 0.90 1.30 Glu 292 A A . . . . .1.90 −1.50 . . F 0.90 4.83 Glu 293 A A . . . . . 2.29 −1.57 . . F 0.903.94 His 294 A A . . . . . 3.07 −1.09 * . F 0.90 3.67 Lys 295 A A . . .. . 3.18 −1.51 . . F 0.90 4.16 His 296 A A . . . . . 3.39 −1.51 * . F0.90 4.70 Thr 297 A A . . . . . 2.58 −1.11 * . F 0.90 5.98 Arg 298 A A .. . . . 2.58 −0.93 * . F 0.90 2.47 Arg 299 A A . . . . . 2.61 −0.93 * .F 0.90 3.14 Gln 300 A A . . . . . 1.68 −1.43 * * F 0.90 3.63 Leu 301 A A. . . . . 1.82 −1.23 * * F 0.90 1.30 Glu 302 . A B . . . . 1.32−1.23 * * F 0.90 1.30 Asp 303 . A B . . . . 1.21 −0.54 * * F 0.75 0.62Ile 304 . A B . . . . 0.76 −0.94 * * F 1.11 1.25 Arg 305 . A B . . . .0.76 −1.20 . * F 1.17 0.72 Leu 306 . A B . . . . 1.36 −0.80 * * F 1.380.69 Asp 307 . . . . T T . 0.47 −0.37 * * F 2.24 1.52 Gly 308 . . . . .T C 0.47 −0.37 . * F 2.10 0.54 Asn 309 . . . . . T C 0.54 0.03 * * F1.44 1.06 Pro 310 . . . . . T C 0.13 0.03 * * F 1.08 0.52 Ile 311 . . B. . . . 0.13 0.41 . . F 0.17 0.71 Asn 312 . . B . . . . −0.57 0.67 . . .−0.19 0.36 Leu 313 . . B . . . . −0.43 1.06 . * . −0.40 0.20 Ser 314 . .B . . . . −0.73 1.06 . * . −0.40 0.45 Leu 315 . . B . . . . −1.11 0.76. * . −0.40 0.37 Phe 316 . . B . . T . −0.47 0.86 . * . −0.20 0.46 Pro317 . . . . T T . −1.17 0.93 . * . 0.20 0.54 Ser 318 . . . . T T . −1.021.33 . * . 0.20 0.56 Ala 319 . . B . . T . −1.53 1.21 . . . −0.20 0.35Tyr 320 . . B . . . . −0.93 1.11 * . . −0.40 0.19 Phe 321 . . B . . . .−0.12 1.11 * . . −0.40 0.21 Cys 322 . . B . . . . −0.72 0.73 * . . −0.400.42 Leu 323 . . B . . . . −0.63 0.91 . * . −0.40 0.22 Pro 324 . . B . .. . −0.93 0.59 . * . −0.40 0.39 Arg 325 . . B B . . . −1.03 0.49 . . .−0.60 0.51 Leu 326 . . B B . . . −0.22 0.34 . . . −0.30 0.61 Pro 327 . .. B T . . −0.26 −0.34 . * . 0.70 0.78 Ile 328 . . . B T . . 0.24 0.01 .. . 0.10 0.34 Gly 329 . . B B . . . 0.07 0.50 . * F −0.60 0.60 Arg 330 .. B B . . . −0.43 0.24 . * F −0.15 0.50 Phe 331 . . B B . . . −0.010.24 * . . −0.30 0.91 Thr 332 . . B B . . . −0.19 −0.01 * * . 0.45 1.17

TABLE V Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . A B .. . . −1.30 0.70 . . . −0.60 0.39 Leu 2 . A B . . . . −1.72 0.96 . . .−0.60 0.25 Leu 3 . A B . . . . −2.14 1.21 . . . −0.60 0.16 Pro 4 . A B .. . . −2.06 1.47 . . . −0.60 0.14 Leu 5 . A B . . . . −1.97 1.24 * . .−0.60 0.22 Leu 6 . A B . . . . −2.18 0.94 . . . −0.60 0.36 Leu 7 . A B .. . . −2.18 0.94 . . . −0.60 0.19 Ser 8 . A B . . . . −1.71 1.20 * . .−0.60 0.19 Ser 9 . . B B . . . −1.84 0.94 . . F −0.45 0.23 Leu 10 . . BB . . . −1.33 0.69 . . F −0.45 0.27 Leu 11 . . . B . . C −0.52 0.39 * .F 0.05 0.27 Gly 12 . . . . . T C −0.30 0.40 . . F 0.45 0.35 Gly 13 . . .. . T C −0.60 0.51 . . F 0.15 0.43 Ser 14 . . B . . T . −0.30 0.44 . . F0.12 0.52 Gln 15 . . B . . T . 0.17 −0.24 . * F 1.19 0.88 Ala 16 . . B .. . . 1.09 −0.24 . * F 1.16 0.88 Met 17 . . B . . T . 0.73 −0.67 . * F1.98 1.28 Asp 18 . . B . . T . 0.79 −0.27 . * F 1.70 0.64 Gly 19 . . . .T T . 0.20 0.24 * * F 1.33 0.67 Arg 20 . . . . T T . 0.31 0.43 * * .0.71 0.47 Phe 21 . . B B . . . 0.04 −0.19 * * . 0.64 0.56 Trp 22 . . B B. . . 0.64 0.46 * * . −0.43 0.42 Ile 23 . . B B . . . 0.64 0.43 * * .−0.60 0.37 Arg 24 . . B B . . . 0.69 0.43 * * . −0.60 0.74 Val 25 . . BB . . . −0.28 0.03 * * . −0.30 0.94 Gln 26 . . . B . . C −0.18 −0.24 * *F 0.65 0.99 Glu 27 . . . B . . C −0.74 −0.31 * * F 0.65 0.50 Ser 28 . .. B T . . −0.07 0.33 . * . 0.10 0.50 Val 29 . . B B . . . −0.18 0.11 . *. −0.30 0.45 Met 30 . . B B . . . 0.09 −0.29 . . . 0.30 0.45 Val 31 . .B B . . . −0.58 0.21 . . . −0.30 0.34 Pro 32 . . B B . . . −0.58 0.40 .. . −0.30 0.24 Glu 33 . . B . . . . −1.17 −0.24 . . . 0.50 0.41 Ala 34 .. . . T . . −0.61 −0.17 . . . 0.90 0.39 Cys 35 . . B . . . . −0.87−0.43 * . . 0.50 0.34 Asp 36 . . . B T . . −0.22 −0.21 * . . 0.70 0.14Ile 37 . . B B . . . −0.68 0.21 . . . −0.30 0.22 Ser 38 . . B B . . .−0.98 0.29 . * . −0.30 0.22 Val 39 . . B . . T . −1.09 0.10 . * . 0.100.18 Pro 40 . . B . . T . −0.72 0.89 . * . −0.20 0.22 Cys 41 . . . . T T. −0.97 0.59 . * . 0.20 0.22 Ser 42 . . . . T T . −0.29 0.96 . * . 0.200.46 Phe 43 . . . . T . . 0.12 0.74 * . . 0.28 0.46 Ser 44 . . B . . . .0.98 0.31 . . . 0.61 1.69 Tyr 45 . . B . . T . 1.19 0.14 . . . 1.09 2.19Pro 46 . . . . T T . 1.57 −0.24 . . F 2.52 4.22 Arg 47 . . . . T T .1.56 −0.11 . . F 2.80 3.31 Gln 48 . . . . T T . 1.91 −0.01 . . F 2.523.05 Asp 49 . . . . T . . 1.91 −0.34 . * F 2.04 1.95 Trp 50 . . . . T T. 1.84 −0.39 . * F 1.96 1.33 Thr 51 . . . . . T C 1.84 0.10 . * F 0.881.11 Gly 52 . . . . T T . 1.14 0.13 . * F 0.80 1.03 Ser 53 . . . . . T C0.90 0.63 . . F 0.15 0.99 Thr 54 . . . . . . C 0.56 0.47 . . F 0.10 1.07Pro 55 . . . . . T C 0.60 0.41 . . F 0.30 1.07 Ala 56 . . . . T T . 0.620.74 . . . 0.35 1.26 Tyr 57 . . . . T T . 0.27 1.27 . . . 0.20 0.91 Gly58 . . . . T T . 0.61 1.57 . . . 0.20 0.51 Tyr 59 . . B B . . . 0.331.14 * . . −0.45 1.01 Trp 60 . . B B . . . −0.31 1.14 * . . −0.60 0.65Phe 61 . . B B . . . −0.03 1.03 * . . −0.60 0.49 Lys 62 . . B B . . .0.21 1.09 * . . −0.60 0.45 Ala 63 . . B B . . . 0.24 0.33 * . . −0.300.74 Val 64 . . B B . . . 0.18 −0.10 * . . 0.79 1.24 Thr 65 . . B B . .. 0.51 −0.40 * . F 1.13 0.89 Glu 66 . . B B . . . 0.87 −0.40 * . F 1.621.77 Thr 67 . . . B T . . 0.23 −0.47 * . F 2.36 2.36 Thr 68 . . . . T T. 0.61 −0.61 * . F 3.40 1.65 Lys 69 . . . . T T . 0.61 −0.67 * . F 3.061.48 Gly 70 . . . . . T C 0.33 −0.03 . . F 2.07 0.76 Ala 71 . . . . . TC 0.02 −0.01 * . F 1.73 0.53 Pro 72 . . B . . . . 0.33 −0.01 * . . 0.840.38 Val 73 . . B . . . . 0.61 0.39 . . . −0.10 0.62 Ala 74 . . B . . .. 0.57 0.46 . . . −0.10 0.84 Thr 75 . . B . . . . 0.61 0.36 * . . 0.500.94 Asn 76 . . . . . . C 1.31 0.31 . . F 1.30 1.70 His 77 . . . . . T C1.52 −0.33 * . F 2.40 3.29 Gln 78 . . . . . T C 1.52 −0.83 * . F 3.003.95 Ser 79 . . . . . T C 2.11 −0.67 * . F 2.70 1.82 Arg 80 . . B . . T. 1.82 −1.07 * . F 2.20 2.32 Glu 81 . A B . . . . 1.52 −0.96 * . F 1.501.33 Val 82 . A B . . . . 1.24 −0.97 * * F 1.54 1.33 Glu 83 . A B . . .. 1.36 −0.87 * * . 1.28 0.98 Met 84 . A B . . . . 1.31 −0.87 * * . 1.771.10 Ser 85 . . . . . T C 1.31 −0.44 * * F 2.56 1.47 Thr 86 . . . . T T. 0.61 −1.09 . * F 3.40 1.67 Arg 87 . . . . T T . 1.47 −0.30 . * F 2.761.46 Gly 88 . . . . T T . 0.66 −0.51 . * F 2.72 1.88 Arg 89 . . B B . .. 0.94 −0.21 . * F 1.28 1.08 Phe 90 . . B B . . . 0.90 −0.21 . * . 0.640.79 Gln 91 . . B B . . . 1.21 0.21 . * . −0.30 0.79 Leu 92 . . B B . .. 0.89 −0.21 . * . 0.30 0.68 Thr 93 . . B B . . . 0.64 0.21 . * F 0.341.21 Gly 94 . . . B . . C 0.58 −0.07 . * F 1.33 0.70 Asp 95 . . . . . TC 0.93 −0.47 * * F 2.22 1.71 Pro 96 . . . . . T C 0.93 −0.73 . * F 2.861.17 Ala 97 . . . . T T . 1.08 −0.81 . . F 3.40 1.90 Lys 98 . . . . T T. 1.09 −0.67 . * F 2.91 0.61 Gly 99 . . . . T T . 0.62 −0.29 * . F 2.270.53 Asn 100 . . B . T T . −0.23 −0.03 . * F 1.93 0.43 Cys 101 . . B . .T . −0.91 0.11 * * . 0.44 0.16 Ser 102 . . B . . T . −0.21 0.80 * * .−0.20 0.11 Leu 103 . A B B . . . −0.26 0.37 * * . −0.30 0.14 Val 104 . AB B . . . −0.50 −0.03 * * . 0.30 0.43 Ile 105 . A B B . . . −0.50−0.10 * . . 0.30 0.33 Arg 106 . A B B . . . −0.43 −0.09 * . . 0.30 0.68Asp 107 . A B B . . . −0.13 −0.16 * . . 0.30 0.91 Ala 108 . A B . . . .0.68 −0.40 * . . 0.45 2.25 Gln 109 . A B . . . . 1.53 −1.09 * . . 1.031.92 Met 110 . A . . . . C 2.12 −1.09 . . . 1.51 1.99 Gln 111 . A B . .. . 2.01 −0.70 . . F 1.74 2.64 Asp 112 . . . . T T . 1.77 −0.80 . * F2.82 2.64 Glu 113 . . . . T T . 1.66 −0.44 * . F 2.80 4.18 Ser 114 . . .. T T . 0.96 −0.27 * * F 2.52 2.09 Gln 115 . . . . T T . 1.67 0.11 * * F1.64 1.08 Tyr 116 . . B B . . . 0.81 0.11 * * . 0.41 1.22 Phe 117 . . BB . . . 0.81 0.76 * . . −0.32 0.68 Phe 118 . . B B . . . 0.92 0.37 * * .0.04 0.68 Arg 119 . . B B . . . 0.88 −0.03 * . . 0.98 0.85 Val 120 . . BB . . . 0.58 −0.36 * . . 1.32 0.97 Glu 121 . . . . T T . 0.58 −0.76 * .F 3.06 1.50 Arg 122 . . . . T T . 0.42 −0.79 * . F 3.40 1.20 Gly 123 . .. . T T . 1.23 −0.14 * * F 2.76 1.20 Ser 124 . . . . T T . 0.88−0.79 * * F 2.72 1.36 Tyr 125 . . B . . . . 1.73 −0.03 . * . 1.33 1.09Val 126 . . B . . . . 1.03 0.37 . * . 0.39 1.76 Arg 127 . . B . . . .0.32 0.73 . . . −0.25 1.14 Tyr 128 . . B . . . . 0.67 0.96 . * . −0.400.72 Asn 129 . . B . . . . 0.97 0.60 . * . −0.25 1.56 Phe 130 . . B . .. . 0.87 −0.04 . * . 0.65 1.33 Met 131 . . B . . . . 1.02 0.39 . * .−0.10 0.84 Asn 132 . . . . T T . 0.21 0.41 . * . 0.20 0.45 Asp 133 . . .. T T . −0.36 0.80 . . . 0.20 0.45 Gly 134 . . . . T T . −0.31 0.70 . *. 0.20 0.38 Phe 135 . . B . . T . −0.47 0.09 . . . 0.10 0.47 Phe 136 . .B B . . . −0.18 0.33 . * . −0.30 0.21 Leu 137 . . B B . . . −1.03 0.81 .. . −0.60 0.30 Lys 138 . . B B . . . −1.84 1.03 . . . −0.60 0.26 Val 139. . B B . . . −1.80 0.93 . . . −0.60 0.25 Thr 140 . . B B . . . −1.800.53 . * . −0.60 0.40 Val 141 . . B B . . . −1.41 0.63 . . . −0.60 0.17Leu 142 . . B B . . . −0.81 1.11 . * . −0.60 0.34 Ser 143 . . B B . . .−0.74 0.90 . * . −0.60 0.36 Phe 144 . . B B . . . −0.10 0.41 * * . −0.260.96 Thr 145 . . . . . T C 0.21 0.20 * * F 1.28 1.80 Pro 146 . . . . . TC 1.07 −0.09 * * F 2.22 2.32 Arg 147 . . . . . T C 1.84 −0.47 . * F 2.564.48 Pro 148 . . . . T T . 2.14 −0.76 . * F 3.40 4.22 Gln 149 . . . . T. . 2.53 −0.84 * * F 2.86 4.39 Asp 150 . . . . T . . 2.84 −0.79 * . F2.52 3.24 His 151 . . . . T . . 2.24 −0.79 * . F 2.18 3.50 Asn 152 . . .. T T . 1.82 −0.53 * * F 2.04 1.67 Thr 153 . . . . T T . 1.37 −0.44 . .F 1.40 1.44 Asp 154 . . . . T T . 1.33 0.13 . * F 0.65 0.57 Leu 155 . .B . . T . 0.48 0.13 * * . 0.10 0.48 Thr 156 . . B B . . . 0.51 0.37 . *. −0.30 0.25 Cys 157 . . B B . . . −0.19 −0.11 . * . 0.49 0.25 His 158 .. B B . . . −0.18 0.67 * * . −0.22 0.26 Val 159 . . B B . . . −0.070.37 * * . 0.27 0.24 Asp 160 . . B B . . . 0.79 −0.11 * * . 1.06 0.88Phe 161 . . B . . . . 0.76 −0.69 * . . 1.90 1.29 Ser 162 . . . . T T .0.57 −0.76 * . F 2.46 1.72 Arg 163 . . . . T T . 0.30 −0.76 * . F 2.120.77 Lys 164 . . . . T T . 0.57 −0.37 * . F 1.78 1.18 Gly 165 . . . . TT . 0.57 −0.66 * . F 1.74 0.89 Val 166 . . . B . . C 1.38 −0.64 * . F0.95 0.79 Ser 167 . . . B . . C 1.37 −0.64 * . F 0.95 0.77 Ala 168 . . BB . . . 0.40 −0.16 * . F 0.60 1.13 Gln 169 . . B B . . . 0.47 0.06 * * F0.00 1.13 Arg 170 . . B B . . . 0.00 −0.59 . * F 0.90 1.65 Thr 171 . . BB . . . 0.97 −0.29 . * F 0.60 1.35 Val 172 . . B B . . . 0.41 −0.79 * *. 0.75 1.52 Arg 173 . . B B . . . 0.41 −0.54 * * . 0.60 0.58 Leu 174 . .B B . . . 0.17 −0.04 * * . 0.30 0.40 Arg 175 . . B B . . . −0.530.23 * * . −0.30 0.85 Val 176 . . B B . . . −0.43 0.09 * * . −0.30 0.44Ala 177 . . B B . . . 0.53 0.51 * * . −0.60 0.82 Tyr 178 . . B B . . .0.42 −0.17 * * . 0.30 0.82 Ala 179 . . B . . T . 0.42 −0.17 * * . 0.851.85 Pro 180 . . B . . T . −0.54 −0.13 * * . 0.85 1.51 Arg 181 . . B . .T . −0.58 0.01 * . F 0.25 0.72 Asp 182 . . B . . T . −0.29 −0.06 * . F0.85 0.50 Leu 183 . . B B . . . −0.93 −0.17 * . . 0.30 0.43 Val 184 . .B B . . . −0.64 0.09 * . . −0.30 0.15 Ile 185 . . B B . . . −0.32 0.47 *. . −0.26 0.12 Ser 186 . . B B . . . −0.43 0.47 * . . 0.08 0.29 Ile 187. . B B . . . −0.43 −0.21 * . . 1.32 0.66 Ser 188 . . B . . T . 0.07−0.46 * . F 2.36 1.52 Arg 189 . . . . T T . 0.71 −0.66 * . F 3.40 1.63Asp 190 . . . . T T . 1.01 −0.61 * . F 3.06 3.61 Asn 191 . . . . . T C0.50 −0.80 * . F 2.52 2.72 Thr 192 . . . . . . C 1.39 −0.50 * . F 1.981.14 Pro 193 . . . . . . C 1.48 −0.50 . . F 1.64 1.19 Ala 194 . . . . T. . 1.37 −0.07 * . F 1.20 1.14 Leu 195 . . B . . . . 1.16 −0.07 . . F1.14 1.37 Glu 196 . . B . . . . 1.16 −0.13 . * F 1.48 1.37 Pro 197 . . B. . . . 1.12 −0.16 . * F 1.82 2.35 Gln 198 . . . . . . C 1.33 −0.23 . *F 2.36 2.82 Pro 199 . . . . T T . 1.07 −0.51 . * F 3.40 2.62 Gln 200 . .. . T T . 1.67 0.13 . * F 2.16 1.26 Gly 201 . . . . T T . 1.42 0.13 . *F 1.82 1.12 Asn 202 . . . . . T C 0.82 0.49 . * F 0.98 1.14 Val 203 . .B . . . . 0.82 0.74 . * F 0.09 0.54 Pro 204 . A B . . . . 0.44 0.34 . .. −0.30 0.95 Tyr 205 . A B . . . . 0.44 0.41 . . . −0.60 0.59 Leu 206 .A B . . . . 0.83 0.41 . . . −0.17 1.39 Glu 207 . A B . . . . 0.49 −0.23. . . 1.01 1.79 Ala 208 . A B . . . . 1.34 −0.23 . . F 1.44 1.13 Gln 209. . B . . T . 0.86 −0.59 . . F 2.42 2.38 Lys 210 . . . . T T . 0.29−0.49 * . F 2.80 1.19 Gly 211 . . . . T T . 1.21 0.20 * . F 1.77 0.97Gln 212 . . B . . T . 0.40 −0.30 * . F 1.84 1.10 Phe 213 . A B . . . .0.18 −0.01 * . . 0.86 0.45 Leu 214 . A B . . . . −0.49 0.67 * . . −0.320.38 Arg 215 . A B . . . . −1.12 0.81 * . . −0.60 0.12 Leu 216 . A B . .. . −1.37 0.91 * . . −0.60 0.14 Leu 217 . A B . . . . −1.37 0.63 * . .−0.60 0.17 Cys 218 . A B . . . . −0.97 −0.06 * * . 0.54 0.14 Ala 219 . A. . T . . −0.16 0.33 * * . 0.58 0.23 Ala 220 . . . . T T . −0.480.04 * * F 1.37 0.49 Asp 221 . . . . T T . 0.12 −0.21 . . F 2.36 1.40Ser 222 . . . . . T C 0.34 −0.36 . . F 2.40 2.15 Gln 223 . . . . . T C0.70 −0.36 . . F 2.16 2.15 Pro 224 . . . . . T C 0.48 −0.37 . . F 1.921.85 Pro 225 . . . . T T . 0.77 0.31 . * F 1.28 1.14 Ala 226 . . . . T T. 0.48 0.31 . . F 0.89 0.88 Thr 227 . . B . . T . −0.08 0.83 . . . −0.200.60 Leu 228 . . B B . . . −0.89 1.04 * * . −0.60 0.29 Ser 229 . . B B .. . −0.68 1.30 . . . −0.60 0.24 Trp 230 . . B B . . . −0.47 1.20 . * .−0.60 0.28 Val 231 . . B B . . . 0.23 1.11 . * . −0.60 0.55 Leu 232 . .B B . . . −0.31 0.43 . * . −0.60 0.80 Gln 233 . . B B . . . −0.31 0.69. * F −0.45 0.57 Asn 234 . . B B . . . −0.31 0.46 . * F −0.45 0.63 Arg235 . . B B . . . −0.32 0.20 . . F 0.00 1.03 Val 236 . . B B . . . 0.23−0.10 . . F 0.45 0.79 Leu 237 . . B . . T . 1.01 −0.11 * * F 0.85 0.66Ser 238 . . B . . T . 0.80 −0.01 * . F 0.85 0.46 Ser 239 . . . . T T .0.51 0.41 * . F 0.35 0.96 Ser 240 . . . . . T C 0.06 0.69 * . F 0.301.22 His 241 . . . . . T C 0.70 0.43 * . F 0.15 0.90 Pro 242 . . . . T T. 1.62 0.47 * . . 0.35 1.04 Trp 243 . . . . T T . 1.71 0.09 * . . 0.651.52 Gly 244 . . . . . T C 1.20 0.13 * . F 0.60 1.73 Pro 245 . . . . . .C 1.16 0.31 . . F 0.25 0.92 Arg 246 . . . . . T C 0.38 0.31 . . F 0.450.87 Pro 247 . . . . . T C 0.59 0.09 * . F 0.45 0.72 Leu 248 . . B . . T. 0.07 −0.34 * . . 0.70 0.81 Gly 249 . . B . . T . 0.20 −0.09 * . . 0.700.34 Leu 250 . . B . . . . 0.07 0.34 * . . −0.10 0.34 Glu 251 . . B . .. . −0.90 0.34 * . . −0.10 0.41 Leu 252 . . B . . . . −0.64 0.30 . . .−0.10 0.31 Pro 253 . . B . . . . −0.42 −0.13 . . F 0.65 0.74 Gly 254 . .B . . . . −0.42 −0.31 . . F 0.65 0.43 Val 255 . . B . . . . 0.39 0.11 *. F 0.05 0.52 Lys 256 . . B . . . . 0.09 −0.57 . . F 1.29 0.56 Ala 257 .. B . . . . 0.56 −0.61 * * F 1.63 0.76 Gly 258 . . . . T . . 0.88−0.61 * * F 2.52 1.02 Asp 259 . . . . T T . 0.98 −1.26 * * F 2.91 0.99Ser 260 . . . . T T . 1.52 −0.50 * * F 3.40 1.54 Gly 261 . . . . T T .0.81 −0.51 * * F 3.06 2.25 Arg 262 . . B . . T . 1.51 −0.37 * * F 1.870.72 Tyr 263 . . B B . . . 1.27 −0.37 * * F 1.28 1.05 Thr 264 . . B B .. . 1.27 −0.26 * * . 0.79 1.08 Cys 265 . . B B . . . 1.57 −0.69 * * .0.94 0.95 Arg 266 . . B B . . . 2.02 −0.29 * * . 0.98 0.98 Ala 267 . . B. . . . 1.10 −1.04 * * F 2.12 1.33 Glu 268 . . B . . . . 1.00 −0.84 * *F 2.46 2.04 Asn 269 . . . . T T . 1.01 −0.99 * * F 3.40 1.03 Arg 270 . .. . T T . 1.68 −0.60 * * F 3.06 1.37 Leu 271 . . . . T T . 1.57−0.70 * * F 2.72 1.37 Gly 272 . . . . T T . 2.27 −0.30 * * F 2.08 1.47Ser 273 . . . . . . C 1.68 −0.70 * * F 1.64 1.47 Gln 274 . A B . . . .0.87 −0.20 * * F 0.60 1.80 Gln 275 . A B . . . . 0.76 −0.20 . * F 0.601.50 Arg 276 . A B . . . . 0.76 −0.63 * . F 0.90 1.87 Ala 277 . A B . .. . 0.80 −0.33 * . F 0.45 0.89 Leu 278 . A B . . . . 0.24 −0.34 * * .0.30 0.69 Asp 279 . A B . . . . 0.24 −0.10 * * . 0.30 0.26 Leu 280 . A B. . . . 0.00 0.30 * * . −0.30 0.45 Ser 281 . . B . . . . −0.32 0.56 * *. −0.40 0.85 Val 282 . . B . . . . 0.06 0.30 * * . −0.10 0.79 Gln 283 .. B . . . . 0.87 0.73 . * . −0.25 1.48 Tyr 284 . . B . . . . 0.87 0.04. * . 0.25 1.91 Pro 285 . . . . . T C 0.87 0.06 * * F 1.00 4.14 Pro 286. . . . . T C 1.28 0.10 * * F 1.20 1.97 Glu 287 . . . . T T . 1.28−0.30 * * F 2.20 2.47 Asn 288 . . B . . T . 0.68 −0.41 * * F 2.00 2.18Leu 289 . . B B . . . 0.07 −0.23 * * . 1.10 0.76 Arg 290 . . B B . . .−0.02 −0.01 * * . 0.90 0.32 Val 291 . . B B . . . 0.19 0.37 * * . 0.100.27 Met 292 . . B B . . . −0.40 0.37 * * . −0.10 0.57 Val 293 . . B B .. . −0.40 0.19 * * . −0.30 0.29 Ser 294 . . B . . . . 0.52 0.59 * * .−0.40 0.63 Gln 295 . . B . . . . 0.10 −0.06 * * F 0.80 1.26 Ala 296 . .B . . . . 0.10 −0.19 * . F 0.80 2.44 Asn 297 . . B . . . . −0.11 −0.19 *. F 0.80 1.35 Arg 298 . . B . . . . 0.74 0.11 * . F 0.05 0.64 Thr 299 .. B . . . . 1.04 −0.29 * . F 0.80 1.10 Val 300 . . B . . . . 0.23−0.39 * . . 0.65 1.10 Leu 301 . . B . . . . 0.48 −0.10 * . . 0.50 0.47Glu 302 . . B . . . . 0.48 0.33 * . . 0.03 0.32 Asn 303 . . B . . . .0.02 0.24 * . F 0.31 0.69 Leu 304 . . . . T T . 0.02 0.03 * . F 1.040.83 Gly 305 . . . . T T . 0.58 −0.17 * . F 1.77 0.69 Asn 306 . . . . TT . 0.58 0.21 * . F 1.30 0.58 Gly 307 . . . . . T C 0.37 0.50 * . F 0.670.58 Thr 308 . . . . . . C −0.49 0.24 . . F 0.64 0.90 Ser 309 . . B . .. . −0.49 0.46 . . F 0.01 0.42 Leu 310 . . B . . . . −0.14 0.74 . . .−0.27 0.35 Pro 311 . . B . . . . −0.49 0.31 . . . −0.10 0.42 Val 312 . .B . . . . −0.14 0.26 . . . −0.10 0.31 Leu 313 . . B . . . . −0.13 0.27 .. F 0.05 0.64 Glu 314 . . B . . . . −0.64 −0.03 . . F 0.65 0.56 Gly 315. . . . T T . −0.50 0.23 . . F 0.65 0.62 Gln 316 . . . . T T . −1.100.16 . . F 0.65 0.40 Ser 317 . . . . T T . −1.10 0.16 . . F 0.65 0.19Leu 318 . . B . . T . −0.96 0.80 . . . −0.20 0.14 Cys 319 . . B B . . .−1.81 0.94 . . . −0.60 0.04 Leu 320 . . B B . . . −1.78 1.19 . . . −0.600.02 Val 321 . . B B . . . −1.81 1.29 . . . −0.60 0.04 Cys 322 . . B B .. . −1.81 1.10 . . . −0.60 0.11 Val 323 . . B B . . . −1.30 0.91 . . .−0.60 0.18 Thr 324 . . B B . . . −0.84 0.61 . . . −0.60 0.32 His 325 . .. . T T . −0.24 0.40 . . F 0.89 0.93 Ser 326 . . . . . T C 0.02 0.26 * *F 1.08 1.93 Ser 327 . . . . . T C 0.80 0.11 * * F 1.32 1.35 Pro 328 . .. . . T C 0.84 −0.37 . * F 2.16 1.95 Pro 329 . . . . T . . 0.86 −0.19. * F 2.40 1.20 Ala 330 . . . . T . . 0.60 −0.19 . * F 2.16 1.20 Arg 331. . B B . . . 0.59 0.34 . * . 0.42 0.82 Leu 332 . . B B . . . 0.89 0.40. * . 0.18 0.76 Ser 333 . . B B . . . 1.21 0.37 . * . 0.09 1.30 Trp 334. . B B . . . 1.08 −0.13 . * . 0.45 1.30 Thr 335 . . B B . . . 1.67 0.30. * F 0.00 1.56 Gln 336 . . B . . T . 0.70 0.01 . * F 0.40 2.02 Arg 337. . B . . T . 0.70 0.27 . * F 0.40 1.43 Gly 338 . . . . T T . 0.70 0.04. * F 0.65 0.82 Gln 339 . . B . . T . 0.78 −0.06 . * F 0.85 0.63 Val 340. . B . . . . 0.79 −0.03 . . F 0.65 0.50 Leu 341 . . B . . . . 0.79 0.36. . F 0.05 0.67 Ser 342 . . B . . T . 0.47 0.33 . * F 0.55 0.67 Pro 343. . . . T T . 0.51 0.36 . . F 1.40 1.41 Ser 344 . . . . T T . 0.51 0.10. . F 1.70 2.28 Gln 345 . . . . . T C 1.16 −0.59 . . F 2.70 2.85 Pro 346. . . . T . . 1.62 −0.54 . . F 3.00 2.85 Ser 347 . . . . . . C 1.07−0.54 . . F 2.50 2.10 Asp 348 . . . . . T C 0.47 −0.29 . . F 1.95 0.90Pro 349 . . B . . T . 0.77 0.00 . . F 1.45 0.48 Gly 350 . . B . . T .−0.04 −0.43 . . F 1.15 0.62 Val 351 . . B . . T . −0.04 −0.13 * . . 0.700.31 Leu 352 . . B . . . . 0.37 0.30 * . . −0.10 0.31 Glu 353 . . B . .. . −0.49 −0.13 * . . 0.50 0.61 Leu 354 . . B B . . . −0.28 0.09 * * .−0.30 0.61 Pro 355 . . B B . . . −0.79 −0.16 * * F 0.60 1.27 Arg 356 . AB B . . . 0.07 −0.20 * * . 0.30 0.55 Val 357 . A B B . . . 0.84 −0.20. * . 0.45 1.15 Gln 358 . A B B . . . 0.84 −0.39 . * . 0.45 1.01 Val 359. A B B . . . 1.31 −0.81 . * . 0.60 0.89 Glu 360 . A B B . . . 1.52−0.39 . * . 0.45 1.19 His 361 . A . . . . C 0.71 −1.03 . * F 1.10 1.19Glu 362 . A . . T . . 1.26 −0.64 . * F 1.30 1.39 Gly 363 . A . . T . .0.59 −0.80 . * F 1.30 1.16 Glu 364 . A . . T . . 1.41 −0.23 * * F 0.850.46 Phe 365 . A . . T . . 0.82 −0.23 * * . 0.70 0.36 Thr 366 A A . . .. . 0.97 0.27 . * . −0.30 0.37 Cys 367 . A . . T . . 0.93 −0.16 * * .0.70 0.41 His 368 . A B . . . . 1.07 0.34 * * . −0.30 0.65 Ala 369 . A .. T . . 0.26 −0.01 * * . 0.95 0.70 Arg 370 . A . . . . C 0.61 0.19 * * .0.55 1.07 His 371 . . . . . T C 0.62 0.04 . * . 1.05 0.78 Pro 372 . . .. T T . 1.29 −0.07 . * . 2.25 1.03 Leu 373 . . . . T T . 1.29 −0.17 . *F 2.50 0.91 Gly 374 . . . . T T . 1.02 0.33 . * F 1.65 0.91 Ser 375 . .B B . . . 0.61 0.47 . * F 0.30 0.44 Gln 376 . . B B . . . −0.17 0.43 . .F 0.05 0.71 His 377 . . B B . . . −0.26 0.43 . * . −0.35 0.59 Val 378 .. B B . . . −0.26 0.39 . * . −0.30 0.59 Ser 379 . . B B . . . −0.21 0.69. * . −0.60 0.28 Leu 380 . . B B . . . −0.77 0.67 . * . −0.60 0.28 Ser381 . . B B . . . −0.80 0.81 . * . −0.60 0.28 Leu 382 . . B B . . .−1.01 0.67 . * . −0.60 0.28 Ser 383 . . B B . . . −0.46 1.04 . * . −0.600.54 Val 384 . . B B . . . −0.37 0.74 . * . −0.60 0.54 His 385 . . B B .. . 0.49 0.79 * * . −0.45 1.01 Tyr 386 . . B B . . . −0.02 0.10 * * .−0.15 1.50 Ser 387 . . B . . T . −0.02 0.40 . * . 0.25 1.67 Pro 388 . .B . . T . −0.07 0.44 * * F 0.10 1.01 Lys 389 . . . . T T . 0.58 0.37 * *F 0.65 0.64 Leu 390 . . . . T T . 0.31 0.04 * . F 0.65 0.74 Leu 391 . .B . . . . −0.11 0.04 . . F 0.05 0.64 Gly 392 . . . . . T C −0.11 0.19 .. F 0.45 0.17 Pro 393 . . . . . T C −0.19 0.57 . . F 0.15 0.28 Ser 394 .. . . . T C −0.23 0.80 . . F 0.15 0.36 Cys 395 . . . . . T C −0.01 0.11. * . 0.30 0.62 Ser 396 . A . . . . C 0.80 0.19 . * . −0.10 0.41 Trp 397. A B . . . . 0.80 −0.24 . . . 0.30 0.52 Glu 398 . A B . . . . 0.20−0.20 . . . 0.30 0.97 Ala 399 . A . . T . . 0.47 −0.09 . . . 0.70 0.60Glu 400 . A . . T . . 0.47 0.03 . . F 0.10 0.77 Gly 401 . A . . T . .0.47 −0.31 . . . 0.70 0.24 Leu 402 . A . . T . . 0.09 0.07 . . . 0.100.32 His 403 . A . . T . . −0.21 0.14 . . . 0.10 0.10 Cys 404 . . . . TT . 0.08 0.53 . . . 0.20 0.13 Ser 405 . . . . T T . 0.08 0.49 . . . 0.200.22 Cys 406 . . . . T T . −0.17 0.20 . . . 0.50 0.27 Ser 407 . . . . TT . 0.34 0.20 . * F 0.65 0.52 Ser 408 . . . . T . . 0.17 0.01 . . F 0.450.52 Gln 409 . . . . T . . 0.24 0.06 . . F 0.60 1.49 Ala 410 . . . . . .C 0.33 −0.01 . . F 1.24 1.13 Ser 411 . . . . . . C 0.70 0.03 . . F 0.881.30 Pro 412 . . . . . . C 0.19 0.03 . * F 1.12 1.00 Ala 413 . . . . . TC 0.60 0.31 . * F 1.41 0.82 Pro 414 . . . . . T C 0.31 −0.19 * * F 2.401.20 Ser 415 . . . . . T C 0.61 0.34 * * F 1.41 0.82 Leu 416 . . B . . T. 0.10 0.83 * * . 0.52 0.85 Arg 417 . . B B . . . −0.03 1.01 * * . −0.120.45 Trp 418 . . B B . . . 0.56 1.01 . * . −0.36 0.33 Trp 419 . A . B .. C 0.77 0.63 . * . −0.40 0.70 Leu 420 . A . B . . C 0.26 −0.06 . * .0.50 0.62 Gly 421 . A . B . . C 0.26 0.63 . * . −0.40 0.49 Glu 422 . A B. . . . 0.14 0.40 . * F −0.15 0.38 Glu 423 . A B . . . . 0.09 −0.51 . .F 0.75 0.80 Leu 424 . A . . . . C 0.38 −0.77 * . F 0.95 0.80 Leu 425 . A. . . . C 0.89 −0.80 * . F 1.25 0.74 Glu 426 . A . . T . . 0.93 −0.41 .. F 1.45 0.58 Gly 427 . A . . T . . 0.93 −0.03 . . F 1.75 0.94 Asn 428 .. . . T T . 0.93 −0.31 . . F 2.60 1.97 Ser 429 . . . . . T C 1.44 −1.00. . F 3.00 1.89 Ser 430 . . . . . T C 1.56 −0.61 . . F 2.70 2.57 Gln 431. . . . . T C 1.56 −0.26 . . F 2.10 1.38 Asp 432 . A . . . . C 1.04−0.66 * . F 1.70 1.79 Ser 433 . A B . . . . 0.73 −0.40 * . F 0.75 0.99Phe 434 . A B . . . . 0.82 −0.30 . * . 0.30 0.82 Glu 435 . A B . . . .0.82 −0.27 . . . 0.43 0.76 Val 436 . A B . . . . 0.52 0.11 * . F 0.110.76 Thr 437 . . B . . T . −0.07 0.11 . . F 0.79 1.18 Pro 438 . . . . .T C −0.11 −0.17 . . F 1.57 0.69 Ser 439 . . . . T T . 0.38 0.26 . . F1.30 0.92 Ser 440 . . . . . T C 0.09 0.04 . . F 0.97 0.98 Ala 441 . . .. . . C 0.36 0.47 . . F 0.34 0.67 Gly 442 . . . . . T C 0.67 0.54 . . F0.41 0.50 Pro 443 . . . . T T . 0.58 0.56 . . F 0.48 0.61 Trp 444 . . .. . T C 0.58 0.56 * . F 0.15 0.80 Ala 445 . . B . . T . 0.07 0.44 * . F0.10 1.09 Asn 446 . . B . . T . 0.36 0.70 . . F −0.05 0.58 Ser 447 . . B. . T . −0.11 0.66 . * F −0.05 0.74 Ser 448 . . B . . T . 0.07 0.43 . *F −0.05 0.60 Leu 449 . . B . . T . 0.01 0.43 . * . −0.20 0.51 Ser 450 .. B . . . . 0.26 0.46 . * . −0.40 0.38 Leu 451 . . B . . T . −0.56 0.50. * . −0.20 0.28 His 452 . . B . . T . −0.56 0.80 . * . −0.20 0.28 Gly453 . . . . T T . −0.56 0.50 . * . 0.20 0.28 Gly 454 . . . . . T C −0.090.50 . * F 0.15 0.45 Leu 455 . . . . . . C −0.60 0.24 * * F 0.39 0.33Ser 456 . . . . . T C 0.32 0.43 * * F 0.43 0.27 Ser 457 . . . . T T .−0.46 0.00 . * F 1.67 0.54 Gly 458 . . B . . T . 0.00 0.26 . * F 0.810.54 Leu 459 . . B . . T . −0.32 −0.43 * * . 1.40 0.79 Arg 460 . A B . .. . 0.49 −0.24 * * . 0.86 0.32 Leu 461 . A B . . . . 0.20 −0.63 * * .1.02 0.56 Arg 462 . A B . . . . 0.21 −0.56 * * . 0.88 0.68 Cys 463 . A B. . . . 0.56 −0.33 * * . 0.44 0.37 Glu 464 . A B . . . . 0.51 0.07 . * .−0.30 0.71 Ala 465 . A . . T . . 0.37 0.03 * * . 0.10 0.27 Trp 466 . A .. T . . 0.83 0.53 * * . −0.20 0.68 Asn 467 . . B . . T . 0.13 0.39 . . .0.10 0.39 Val 468 . . B . . T . 0.80 0.89 . . . −0.20 0.39 His 469 . . .. . T C 0.50 0.79 . . . 0.00 0.64 Gly 470 . . . . . T C 0.74 0.26 . . .0.30 0.54 Ala 471 . . . . . . C 0.73 0.29 . . F 0.25 0.72 Gln 472 . . .. . T C −0.16 0.03 . . F 0.45 0.71 Ser 473 . . . . . T C −0.11 0.21 . .F 0.45 0.50 Gly 474 . . B . . T . −0.08 0.47 . . F −0.05 0.41 Ser 475 .. B . . T . −0.54 0.37 . . F 0.25 0.41 Ile 476 . . B . . . . −0.17 0.66. . . −0.40 0.25 Leu 477 . . B . . . . −0.17 0.70 * . . −0.06 0.39 Gln478 . . B . . . . 0.18 0.27 * . . 0.58 0.49 Leu 479 . . B . . T . 0.57−0.11 * . . 1.87 1.39 Pro 480 . . B . . T . 0.52 −0.80 * . F 2.66 3.38Asp 481 . . . . T T . 0.60 −1.06 . . F 3.40 1.93 Lys 482 . . . . T T .0.52 −0.77 . . F 3.06 1.93 Lys 483 . . . B T . . 0.22 −0.77 . . F 2.170.88 Gly 484 . . B B . . . 0.72 −0.81 . . F 1.43 0.70 Leu 485 . . B B .. . 0.34 −0.33 . . F 0.79 0.51 Ile 486 . . B B . . . −0.36 0.17 . . .−0.30 0.26 Ser 487 . . B B . . . −0.70 0.96 . . . −0.60 0.22 Thr 488 . .B B . . . −0.74 0.91 * . . −0.60 0.36 Ala 489 . . B B . . . −0.74 0.63 *. . −0.60 0.84 Phe 490 . . B . . T . −0.52 0.37 . . F 0.25 0.62 Ser 491. . . . . T C −0.33 0.49 * . F 0.15 0.43 Asn 492 . . . . . T C −0.840.79 . . F 0.15 0.37 Gly 493 . . . . . T C −0.88 0.97 . . F 0.15 0.35Ala 494 . . . B . . C −1.18 0.61 . . . −0.40 0.26 Phe 495 . . B B . . .−0.82 0.91 . . . −0.60 0.11 Leu 496 . . B B . . . −1.41 0.94 . . . −0.600.11 Gly 497 . . B B . . . −1.72 1.20 . . . −0.60 0.08 Ile 498 . . B B .. . −1.97 1.19 . . . −0.60 0.13 Gly 499 . . B B . . . −2.19 0.90 . . .−0.60 0.16 Ile 500 . A B . . . . −2.30 0.90 . . . −0.60 0.13 Thr 501 . AB . . . . −2.19 1.16 . . . −0.60 0.16 Ala 502 . A B . . . . −2.66 1.26 .. . −0.60 0.14 Leu 503 . A B . . . . −2.43 1.51 . . . −0.60 0.16 Leu 504. A B . . . . −2.90 1.40 . . . −0.60 0.06 Phe 505 . A B . . . . −2.601.60 . . . −0.60 0.05 Leu 506 . A B . . . . −3.10 1.60 . . . −0.60 0.06Cys 507 A A . . . . . −3.40 1.60 . . . −0.60 0.06 Leu 508 A A . . . . .−3.48 1.60 . . . −0.60 0.05 Ala 509 A A . . . . . −3.27 1.50 * . . −0.600.04 Leu 510 A A . . . . . −2.52 1.43 * . . −0.60 0.08 Ile 511 . A B . .. . −2.60 0.86 * . . −0.60 0.19 Ile 512 . A B . . . . −2.74 0.86 * . .−0.60 0.13 Met 513 . A B . . . . −2.14 1.04 * . . −0.60 0.13 Lys 514 . AB . . . . −1.51 0.79 * . . −0.26 0.28 Ile 515 . A B . . . . −0.59 0.10 .. . 0.38 0.81 Leu 516 . A B . . . . 0.41 −0.59 . * . 1.77 1.61 Pro 517 .. . . . T C 0.99 −1.20 . . F 2.86 1.57 Lys 518 . . . . T T . 1.59 −0.71. * F 3.40 3.24 Arg 519 . . . . T T . 1.23 −1.00 . * F 3.06 6.80 Arg 520. . . . T T . 2.12 −1.20 . . F 2.72 6.34 Thr 521 . . . . T . . 2.62−1.63 . . F 2.18 5.49 Gln 522 . . B . . . . 2.62 −1.14 * . F 1.44 4.05Thr 523 . . B . . . . 2.69 −0.71 * . F 1.10 3.20 Glu 524 . . B . . . .2.37 −0.71 * * F 1.10 4.34 Thr 525 . . B . . T . 2.37 −0.77 . * F 1.303.87 Pro 526 . . . . . T C 1.98 −1.17 . * F 1.50 5.26 Arg 527 . . . . .T C 1.68 −0.87 * * F 1.84 2.63 Pro 528 . . . . T T . 2.10 −0.49 * * F2.08 2.44 Arg 529 . . . . T . . 2.07 −0.97 * * F 2.52 3.09 Phe 530 . . .. T . . 2.08 −0.90 * * F 2.86 2.15 Ser 531 . . . . T T . 1.98 −0.51 . *F 3.40 1.86 Arg 532 . . B . . T . 0.98 −0.46 * * F 2.36 1.37 His 533 . .B . . T . 0.38 0.23 * . F 1.42 1.11 Ser 534 . . B . . T . 0.27 0.13 * .F 0.93 0.68 Thr 535 . . B B . . . 0.72 −0.26 * . . 0.64 0.58 Ile 536 . .B B . . . 0.13 0.50 * . . −0.60 0.67 Leu 537 . . B B . . . 0.02 0.69 . *. −0.60 0.35 Asp 538 . . B B . . . −0.80 0.70 * . . −0.60 0.39 Tyr 539 .. B B . . . −1.36 0.86 * . . −0.60 0.41 Ile 540 . . B B . . . −1.260.81 * . . −0.60 0.37 Asn 541 . . B B . . . −0.68 0.56 * . . −0.60 0.34Val 542 . . B B . . . −0.46 1.04 * . . −0.60 0.32 Val 543 . . B B . . .−0.80 0.79 * . . −0.60 0.46 Pro 544 . . B . . . . −0.77 0.53 * . F −0.250.28 Thr 545 . . B . . T . −0.69 0.56 * . F −0.05 0.59 Ala 546 . . B . .T . −1.28 0.60 * . F −0.05 0.65 Gly 547 . . B . . T . −0.42 0.46 * . F−0.05 0.43 Pro 548 . . B . . T . 0.48 0.43 * . F 0.21 0.51 Leu 549 . . B. . . . 0.80 −0.06 . . F 1.32 1.01 Ala 550 . . B . . . . 1.11 −0.56 . .F 1.88 2.00 Gln 551 . . B . . . . 1.70 −0.59 . . F 2.14 2.08 Lys 552 . .B . . T . 2.09 −0.61 * . F 2.60 4.38 Arg 553 . . B . . T . 1.71 −1.30 .. F 2.34 8.66 Asn 554 . . B . . T . 2.21 −1.30 . . F 2.08 5.05 Gln 555 .. B . . T . 2.59 −1.21 * . F 2.10 3.65 Lys 556 . . B . . . . 2.59−0.79 * . F 1.92 2.88 Ala 557 . . . . . . C 2.24 −0.39 * . F 1.84 2.88Thr 558 . . . . . T C 1.92 −0.40 * . F 2.32 2.23 Pro 559 . . . . T T .2.03 −0.37 * . F 2.80 1.72 Asn 560 . . . . T T . 1.72 −0.37 * . F 2.523.34 Ser 561 . . . . . T C 1.47 −0.39 * * F 2.04 3.34 Pro 562 . . . . T. . 1.24 −0.44 * . F 1.76 3.34 Arg 563 . . . . T . . 1.34 −0.19 * . F1.48 1.71 Thr 564 . . B . . . . 1.34 −0.16 * . F 0.80 1.98 Pro 565 . . B. . . . 1.00 −0.11 * . F 0.80 1.98 Leu 566 . . B . . . . 0.71 −0.11 . *F 0.65 1.00 Pro 567 . . B . . T . 0.71 0.39 . * F 0.25 0.70 Pro 568 . .. . T T . 0.30 0.33 . . F 0.65 0.70 Gly 569 . . . . . T C 0.40 0.29 . .F 0.60 1.14 Ala 570 . . . . . T C 0.61 0.03 . . F 0.94 1.14 Pro 571 . .. . . . C 1.12 −0.40 . . F 1.68 1.27 Ser 572 . . . . . T C 1.38 −0.44 .. F 2.22 1.72 Pro 573 . . . . . T C 1.63 −0.87 . . F 2.86 3.41 Glu 574 .. . . T T . 1.98 −1.37 . . F 3.40 4.41 Ser 575 . . . . T T . 2.57 −1.40. . F 3.06 5.29 Lys 576 . . . . T T . 2.82 −1.39 . . F 2.94 5.93 Lys 577. . . . T T . 3.17 −1.81 . . F 2.82 6.84 Asn 578 . . . . T T . 3.38−1.81 . . F 2.70 10.21 Gln 579 . . . . T T . 3.13 −1.80 * . F 2.58 8.84Lys 580 . . B . . . . 3.43 −1.04 * . F 2.20 6.93 Lys 581 . . B . . . .2.58 −0.64 * . F 1.98 7.46 Gln 582 . . B . . . . 2.32 −0.36 * . F 1.463.55 Tyr 583 . . B . . . . 2.02 −0.33 . . . 1.09 2.75 Gln 584 . . B . .. . 1.32 0.06 . . . 0.27 1.84 Leu 585 . . B . . T . 1.07 0.84 . . .−0.20 0.92 Pro 586 . . B . . T . 1.02 0.87 . . F −0.05 0.91 Ser 587 . .B . . T . 0.81 0.11 . . F 0.59 0.91 Phe 588 . . B . . T . 1.10 0.14 . .F 1.08 1.70 Pro 589 . . . . . . C 0.80 −0.54 . . F 2.32 2.20 Glu 590 . .. . . . C 1.31 −0.59 . . F 2.66 2.20 Pro 591 . . . . T T . 1.21 −0.59 .. F 3.40 3.41 Lys 592 . . . . T T . 1.51 −0.89 . . F 3.06 3.18 Ser 593 .. . . . T C 1.62 −0.91 . . F 2.52 3.18 Ser 594 . . . . . T C 1.62−0.41 * . F 1.88 2.08 Thr 595 . . . . . . C 1.62 −0.41 * . F 1.64 1.61Gln 596 . . . . . . C 1.53 −0.41 * . F 1.60 2.08 Ala 597 . . . . . T C1.49 −0.41 * . F 2.10 2.08 Pro 598 . . . . . T C 1.79 −0.40 . . F 2.402.49 Glu 599 . . . . . T C 1.79 −0.89 . . F 3.00 2.49 Ser 600 . . . . .T C 2.10 −0.90 . . F 2.70 3.31 Gln 601 . A . . . . C 2.10 −1.00 . . F2.00 3.70 Glu 602 . A . . . . C 2.69 −1.43 . . F 1.70 3.70 Ser 603 . A .. . . C 2.09 −1.43 . . F 1.40 4.79 Gln 604 A A . . . . . 2.06 −1.13 . .F 0.90 2.28 Glu 605 A A . . . . . 2.11 −1.03 * . F 0.90 1.79 Glu 606 A A. . . . . 1.52 −0.27 * . F 0.60 2.09 Leu 607 . A B . . . . 1.21 −0.16 .. . 0.45 1.22 His 608 . A B . . . . 0.70 −0.07 . . . 0.45 1.02 Tyr 609 .A B . . . . 0.70 0.61 . . . −0.60 0.48 Ala 610 . A B . . . . 0.00 1.01 *. . −0.60 0.95 Thr 611 . A B . . . . −0.21 1.11 . * . −0.60 0.60 Leu 612. A . . T . . 0.26 1.04 . . . −0.20 0.59 Asn 613 . A B . . . . −0.570.71 * . . −0.60 0.58 Phe 614 . . B B . . . −0.21 0.86 . . . −0.60 0.30Pro 615 . . . B T . . 0.17 0.37 . * F 0.25 0.71 Gly 616 . . . B T . .0.59 0.11 . * F 0.25 0.68 Val 617 . . . B . . C 1.19 −0.29 . * F 0.801.54 Arg 618 . . . . . T C 1.19 −0.64 . * F 1.50 1.54 Pro 619 . . . . .T C 1.30 −1.07 . * F 1.50 2.70 Arg 620 . . . . . T C 1.62 −1.00 . * F1.50 3.68 Pro 621 . . B . . T . 1.37 −1.64 . * F 1.30 3.68 Glu 622 . . .. T . . 2.01 −1.03 * * F 1.84 2.35 Ala 623 . . B . . . . 1.94 −1.03 * *F 1.78 1.86 Arg 624 . . B . . . . 1.81 −1.03 * * . 1.97 2.40 Met 625 . .B . . T . 1.39 −1.03 * * F 2.66 1.37 Pro 626 . . . . T T . 1.60 −0.54. * F 3.40 1.96 Lys 627 . . . . T T . 1.01 −0.64 . * F 3.06 1.74 Gly 628. . . . . T C 1.60 −0.14 * . F 2.22 1.77 Thr 629 . A . . . . C 1.24−0.76 * . F 1.78 1.91 Gln 630 . A B . . . . 1.26 −0.43 . . F 0.94 1.50Ala 631 . A B . . . . 1.47 0.07 . . F 0.00 1.53 Asp 632 . A B . . . .0.57 −0.36 . . . 0.45 1.84 Tyr 633 . A B . . . . 0.96 −0.20 . * . 0.300.79 Ala 634 . A B . . . . 0.57 −0.60 . * . 0.75 1.56 Glu 635 . A B . .. . 0.57 −0.31 . * . 0.30 0.81 Val 636 . A B . . . . 0.77 0.09 . * .−0.30 0.89 Lys 637 . A B . . . . 0.38 −0.24 . * . 0.45 1.13 Phe 638 . AB . . . . 0.23 −0.31 . * . 0.30 0.83 Gln 639 . A B . . . . 0.43 0.11 . *. −0.15 1.44

TABLE VI Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . A B. . . . −1.90 0.96 . . . −0.60 0.21 Leu 2 . A B . . . . −2.32 1.21 . . .−0.60 0.13 Leu 3 . A B . . . . −2.74 1.47 . . . −0.60 0.09 Leu 4 . A B .. . . −2.57 1.73 . . . −0.60 0.07 Leu 5 . A B . . . . −2.99 1.54 . . .−0.60 0.14 Leu 6 . A B . . . . −3.20 1.54 . . . −0.60 0.14 Leu 7 . A B .. . . −2.68 1.54 . . . −0.60 0.14 Pro 8 . A B . . . . −2.21 1.77 * . .−0.60 0.17 Leu 9 . A B . . . . −1.29 1.51 . . . −0.60 0.21 Leu 10 . A B. . . . −0.48 0.83 . * . −0.60 0.49 Trp 11 . A . . . . C 0.44 0.14 . * .−0.10 0.55 Gly 12 . A . . . . C 0.40 −0.29 . * F 0.80 1.31 Arg 13 . A .. . . C 0.61 −0.33 . * F 0.80 1.18 Glu 14 . A B . . . . 1.08 −1.01 . * F0.90 1.94 Arg 15 . A B . . . . 1.89 −1.50 . * F 1.24 1.94 Val 16 . A B .. . . 2.22 −1.53 . * F 1.58 1.71 Glu 17 . A . . T . . 2.27 −1.53 . * F2.32 1.98 Gly 18 . . . . T . . 2.16 −1.14 * * F 2.86 1.35 Gln 19 . . . .T T . 2.27 −0.74 . * F 3.40 2.93 Lys 20 . . . . . T C 2.20 −1.39 . * F2.86 3.32 Ser 21 . . . . . T C 3.06 −1.39 . . F 2.86 6.70 Asn 22 . . . .T T . 2.81 −1.81 * . F 3.06 6.46 Arg 23 . . . . T T . 2.86 −1.46 * . F3.06 5.06 Lys 24 . . . . T T . 2.04 −1.07 * . F 3.06 5.06 Asp 25 . . . .T T . 1.69 −0.77 * . F 3.40 2.60 Tyr 26 . . B . . T . 1.39 −0.69 . * .2.51 1.91 Ser 27 . . B B . . . 1.39 −0.07 . * . 1.32 0.95 Leu 28 . . B B. . . 0.98 0.33 . * . 0.38 0.98 Thr 29 . . B B . . . 0.63 0.71 . * .−0.26 0.84 Met 30 . . B . . T . −0.22 0.34 . * . 0.10 0.84 Gln 31 . . B. . T . −0.29 0.60 . * F −0.05 0.76 Ser 32 . . B . . T . −0.84 0.40 . *F 0.25 0.76 Ser 33 . . B . . T . −0.03 0.56 . * F −0.05 0.57 Val 34 . .B B . . . 0.28 0.34 . * F −0.15 0.57 Thr 35 . . B B . . . 0.53 −0.06 . *F 0.45 0.73 Val 36 . . B B . . . −0.07 −0.01 . . F 0.45 0.54 Gln 37 . .B B . . . −0.43 0.21 . . F −0.15 0.72 Glu 38 . . B B . . . −0.99 0.14 .. F −0.15 0.27 Gly 39 . . . B T . . −0.17 0.30 . * . 0.10 0.27 Met 40 .. B B . . . −0.71 0.16 * * . −0.30 0.21 Cys 41 . . B B . . . 0.260.40 * * . −0.30 0.09 Val 42 . . B B . . . −0.41 0.40 * * . −0.30 0.18His 43 . . B B . . . −0.71 0.54 * * . −0.60 0.10 Val 44 . . B B . . .−1.07 0.31 . * . −0.30 0.24 Arg 45 . . B B . . . −0.77 0.53 . * . −0.600.28 Cys 46 . . B . . T . −0.34 0.27 . * . 0.10 0.28 Ser 47 . . . . T T. 0.30 0.53 . * . 0.20 0.59 Phe 48 . . . . T T . −0.52 0.31 * * . 0.500.46 Ser 49 . . B . . T . 0.33 0.96 * * . −0.20 0.64 Tyr 50 . . B . . .. −0.08 0.39 . * . −0.10 0.80 Pro 51 . . B . . T . 0.59 0.39 . * . 0.251.24 Val 52 . . . . T T . 0.58 0.00 * * F 1.40 1.60 Asp 53 . . B . . T .1.28 0.10 * * F 0.74 1.47 Ser 54 . . B . . T . 1.28 −0.66 . * F 1.981.59 Gln 55 . . B . . . . 1.52 −0.70 . * F 2.12 2.87 Thr 56 . . . . T .. 1.52 −1.34 . * F 2.86 2.87 Asp 57 . . . . T T . 1.52 −0.91 . * F 3.403.31 Ser 58 . . . . . T C 1.49 −0.66 . . F 2.86 1.42 Asp 59 . . B . . T. 1.44 −0.56 . . F 2.32 1.34 Pro 60 . . B . . T . 1.20 −0.61 . . F 1.830.79 Val 61 . . . . T . . 1.22 0.14 . . . 0.64 0.93 His 62 . . B . . T .0.52 0.67 * . . −0.20 0.58 Gly 63 . . B . . T . 0.93 1.46 * . . −0.200.33 Tyr 64 . . B . . T . 0.34 1.03 * . . −0.20 0.86 Trp 65 . . B . . T. 0.21 0.89 * . . −0.20 0.64 Phe 66 . . B . . . . 1.07 0.81 * . . −0.090.64 Arg 67 . . B . . T . 1.10 0.79 * * . 0.42 0.66 Ala 68 . . . . T T .0.56 0.03 * * . 1.58 1.04 Gly 69 . . . . T T . 0.50 −0.20 * * F 2.490.85 Asn 70 . . . . T T . 0.50 −0.60 * * F 3.10 0.58 Asp 71 . . . . . .C 1.24 0.31 * * F 1.49 0.60 Ile 72 . . . . . . C 0.54 −0.19 * * F 1.931.22 Ser 73 . A . . T . . 0.92 −0.11 . * . 1.32 0.76 Trp 74 . A B . . .. 0.41 −0.09 . * . 0.61 0.71 Lys 75 . A B . . . . −0.18 0.56 . * . −0.600.75 Ala 76 . A B . . . . −0.49 0.37 . * . −0.30 0.56 Pro 77 . . B . . .. 0.40 0.47 . * . −0.40 0.77 Val 78 . . B . . . . 0.70 −0.04 . * . 0.500.62 Ala 79 . . B . . . . 0.78 0.36 . * . −0.10 0.99 Thr 80 . . . . . .C 0.14 0.29 . . F 0.25 0.99 Asn 81 . . . . . . C 0.44 0.36 . . F 0.401.35 Asn 82 . . . . . T C 0.07 0.63 . . F 0.30 1.41 Pro 83 . . . . . T C0.07 0.63 . . F 0.15 0.98 Ala 84 . . . . T T . 0.66 0.79 * . . 0.20 0.45Trp 85 . . . . . T C 0.97 0.79 . . . 0.00 0.49 Ala 86 . A B . . . . 0.970.39 . . . −0.30 0.55 Val 87 . A B . . . . 0.66 −0.04 * * . 0.30 0.94Gln 88 . A B . . . . 0.98 −0.06 * . F 0.94 1.29 Glu 89 . A B . . . .1.57 −0.97 . * F 1.58 2.50 Glu 90 . A B . . . . 1.97 −1.47 . * F 1.925.62 Thr 91 . . . . T T . 1.86 −2.11 . * F 3.06 6.36 Arg 92 . . . . T T. 2.68 −1.73 . * F 3.40 3.18 Asp 93 . . . . T T . 1.87 −1.23 . * F 3.062.50 Arg 94 . . B . . T . 1.06 −0.54 * * . 2.17 1.43 Phe 95 . A B . . .. 0.71 −0.34 * * . 0.98 0.60 His 96 . A B . . . . 1.02 0.09 * * . 0.040.36 Leu 97 . A . . . . C 0.70 0.09 . * . −0.10 0.30 Leu 98 . A . . T .. 0.70 0.51 . * . −0.20 0.54 Gly 99 . A . . T . . 0.28 0.13 * * F 0.590.69 Asp 100 . . . . . . C 1.02 0.11 * * F 1.08 1.21 Pro 101 . . . . T .. 1.06 −0.57 . . F 2.52 2.93 Gln 102 . . . . T . . 1.20 −0.86 . . F 2.864.76 Thr 103 . . . . T T . 1.70 −0.71 . . F 3.40 1.53 Lys 104 . . B . .T . 1.23 −0.23 . . F 2.36 1.43 Asn 105 . . B . . T . 0.93 0.03 . . F1.27 0.68 Cys 106 . . B . . T . 0.26 0.01 * * . 0.78 0.63 Thr 107 . . BB . . . 0.37 0.21 * * . 0.04 0.22 Leu 108 . . B B . . . 0.68 0.21 * * .−0.30 0.27 Ser 109 . . B B . . . 0.04 −0.19 * . . 0.30 0.84 Ile 110 . .B B . . . 0.16 −0.26 * * . 0.30 0.59 Arg 111 . . B B . . . 0.22−0.74 * * F 0.90 1.39 Asp 112 . A B . . . . 0.23 −0.81 * . F 0.90 1.03Ala 113 . A B . . . . 1.04 −0.81 * . F 0.90 1.97 Arg 114 . A B . . . .0.76 −1.50 * . . 1.03 1.68 Met 115 . A B . . . . 1.30 −1.00 . * F 1.461.01 Ser 116 . A . . . . C 1.30 −0.57 . * F 1.79 0.99 Asp 117 . . . . TT . 1.06 −1.07 * * F 2.67 0.99 Ala 118 . . . . T T . 0.94 −0.31 * * F2.80 1.57 Gly 119 . . . . T T . 0.13 −0.14 * * F 2.52 1.02 Arg 120 . . B. . T . 0.84 0.26 * * . 0.94 0.53 Tyr 121 . . B B . . . 0.54 0.26 * * .0.41 1.02 Phe 122 . . B B . . . 0.54 0.37 * * . 0.13 1.02 Phe 123 . . BB . . . 1.18 −0.06 * * . 0.30 0.90 Arg 124 . . B B . . . 1.18 −0.06 * *. 0.79 1.15 Met 125 . . B . . . . 1.07 −0.39 * * . 1.33 1.32 Glu 126 . .. . T T . 0.42 −0.77 * * F 2.72 2.45 Lys 127 . . . . T T . 1.17−0.87 * * F 2.91 0.88 Gly 128 . . . . T T . 1.58 −0.87 * * F 3.40 1.77Asn 129 . . . . T T . 1.47 −0.57 * * F 3.06 1.08 Ile 130 . . . . . . C1.82 −0.17 . * F 1.87 0.86 Lys 131 . . . . T . . 1.87 0.59 . * . 0.831.37 Trp 132 . . . . T . . 1.58 0.16 . * . 0.79 1.70 Asn 133 . . B . . T. 1.92 0.51 . * . 0.19 3.81 Tyr 134 . . B . . T . 1.92 −0.17 . * . 1.333.18 Lys 135 . . B . . T . 2.00 0.23 * * . 0.97 5.23 Tyr 136 . . . . T T. 1.66 0.00 . * F 2.36 2.68 Asp 137 . . . . T . . 1.09 −0.01 . * F 2.402.30 Gln 138 . . B B . . . 1.09 −0.13 . * F 1.41 0.85 Leu 139 . . B B .. . 0.48 0.27 . * . 0.42 0.87 Ser 140 . . B B . . . 0.12 0.16 . * . 0.180.39 Val 141 . . B B . . . −0.22 0.64 * * . −0.36 0.32 Asn 142 . . B B .. . −1.03 0.74 . * . −0.60 0.40 Val 143 . . B B . . . −1.34 0.74 . * .−0.60 0.24 Thr 144 . . B B . . . −0.57 0.84 * * . −0.60 0.47 Ala 145 . .B B . . . −0.16 0.70 . * . −0.60 0.40 Leu 146 . . B B . . . 0.49 0.30. * . −0.15 1.06 Thr 147 . . B B . . . 0.49 0.09 . * . −0.15 1.14 His148 . . B B . . . 0.46 0.00 . * . 0.45 1.81 Arg 149 . . B . . T . −0.040.19 . * . 0.25 1.54 Pro 150 . . B . . T . −0.34 0.19 . * . 0.10 0.88Asn 151 . . . . T T . 0.26 0.39 . * . 0.50 0.45 Ile 152 . . B . . T .0.22 0.31 . * . 0.10 0.36 Leu 153 . . B . . . . −0.06 0.74 . * . −0.400.23 Ile 154 . . B . . T . −0.98 0.80 * * . −0.20 0.21 Pro 155 . . B . .T . −0.77 1.09 . * F −0.05 0.24 Gly 156 . . B . . T . −1.07 0.40 . * F0.25 0.51 Thr 157 . . B . . T . −0.52 0.10 . . F 0.25 0.97 Leu 158 . . B. . . . −0.38 −0.16 . . F 0.65 0.62 Glu 159 . . B . . T . −0.19 −0.01 .. F 0.85 0.34 Ser 160 . . . . T T . 0.02 0.34 . . F 0.65 0.20 Gly 161 .. . . T T . 0.37 0.26 . . F 0.61 0.42 Cys 162 . . . . T T . −0.13 −0.03. . . 1.02 0.39 Phe 163 . . B B . . . 0.37 0.66 . . . −0.72 0.24 Gln 164. . B B . . . −0.30 0.76 . . . −0.76 0.35 Asn 165 . . . B T . . −0.300.90 * . . −0.40 0.35 Leu 166 . . B B . . . −0.81 0.71 * . . −0.76 0.55Thr 167 . . . B T . . −0.36 0.57 * . . −0.32 0.23 Cys 168 . . . B T . .0.06 0.60 * . . −0.28 0.22 Ser 169 . . . B T . . −0.53 1.11 * . . −0.240.29 Val 170 . . . B . . C −1.20 0.93 * * . −0.40 0.20 Pro 171 . . . . T. . −0.39 1.01 * . . 0.00 0.20 Trp 172 . . . . T . . −0.08 0.44 . . .0.00 0.26 Ala 173 . . B . . . . 0.24 0.46 . . . −0.12 0.61 Cys 174 . . B. . . . 0.23 0.24 . . . 0.46 0.39 Glu 175 . . . . T T . 0.88 0.30 . . F1.49 0.53 Gln 176 . . . . T T . 0.88 −0.19 . . F 2.37 0.81 Gly 177 . . .. T T . 0.57 −0.26 . . F 2.80 2.35 Thr 178 . . . . . T C 0.27 −0.21 . .F 2.32 1.34 Pro 179 . . . . . . C 0.63 0.47 * . F 0.79 0.54 Pro 180 . .. B . . C 0.34 0.46 * . F 0.31 0.74 Met 181 . . B B . . . −0.26 0.94 * .. −0.32 0.54 Ile 182 . . B B . . . −0.26 1.07 . . . −0.60 0.34 Ser 183 .. B B . . . −0.26 1.07 . . . −0.60 0.22 Trp 184 . . B B . . . −0.341.13 * . . −0.60 0.32 Met 185 . . B B . . . −0.99 0.90 * . . −0.60 0.61Gly 186 . . . B T . . −0.69 0.86 * . . −0.20 0.34 Thr 187 . . . B . . C−0.01 0.86 * . F −0.25 0.43 Ser 188 . . . B . . C −0.52 0.37 * . F 0.050.67 Val 189 . . B B . . . −0.27 0.44 . . F −0.45 0.56 Ser 190 . . . . .. C 0.12 0.51 . . F −0.05 0.53 Pro 191 . . . . . . C 0.17 0.46 . . F−0.05 0.61 Leu 192 . . . . . . C 0.17 0.46 . . F 0.34 1.10 His 193 . . B. . T . 0.16 0.30 * . F 0.88 1.19 Pro 194 . . B . . T . 1.12 0.40 * . F0.82 1.11 Ser 195 . . . . T T . 1.12 −0.03 * . F 2.36 2.64 Thr 196 . . .. . T C 1.03 −0.33 . . F 2.40 2.60 Thr 197 . . B . . T . 0.99 −0.44 . .F 1.96 2.25 Arg 198 . . B . . T . 0.21 −0.23 . . F 1.72 1.25 Ser 199 . .B . . T . 0.11 0.07 . . F 0.73 0.71 Ser 200 . . B . . T . −0.40 0.07 . .F 0.49 0.71 Val 201 . . B B . . . −0.98 0.27 . . . −0.30 0.30 Leu 202 .. B B . . . −0.88 0.96 . . . −0.60 0.16 Thr 203 . . B B . . . −0.99 1.00. . . −0.60 0.18 Leu 204 . . B B . . . −0.90 1.01 * . . −0.60 0.42 Ile205 . . B B . . . −0.60 0.80 * . . −0.60 0.79 Pro 206 . . B B . . . 0.220.51 * . F −0.45 0.95 Gln 207 . . B . . . . 1.00 0.53 * . F −0.10 1.57Pro 208 . . B . . . . 0.97 0.34 * . F 0.20 3.04 Gln 209 . . . . T . .1.47 0.09 * . F 0.60 1.95 His 210 . . . . T T . 2.06 0.14 * . F 0.801.62 His 211 . . . . T T . 1.46 0.13 . . F 0.80 1.41 Gly 212 . . . . T T. 1.14 0.39 . . F 0.65 0.67 Thr 213 . . . . T T . 0.69 0.47 . . F 0.350.71 Ser 214 . . . B T . . 0.69 0.54 * . F −0.05 0.28 Leu 215 . . B B .. . −0.13 0.44 * . . −0.60 0.49 Thr 216 . . B B . . . −0.41 0.66 . * .−0.60 0.25 Cys 217 . . B B . . . −0.88 0.66 . * . −0.60 0.27 Gln 218 . .B B . . . −0.78 0.96 . * . −0.60 0.27 Val 219 . . B B . . . −0.82 0.70. * . −0.60 0.29 Thr 220 . . B B . . . −0.60 0.64 . * . −0.60 0.54 Leu221 . . B . . T . −0.63 0.57 . . . −0.20 0.31 Pro 222 . . B . . T .−0.82 0.60 . . F −0.05 0.42 Gly 223 . . . . T T . −1.13 0.60 . . F 0.350.21 Ala 224 . . . . . T C −0.59 0.60 . . F 0.35 0.38 Gly 225 . . B . .. . −0.28 0.40 * . F 0.15 0.35 Val 226 . . B . . . . 0.64 0.37 * . F0.65 0.57 Thr 227 . . B . . T . 0.54 −0.06 * . F 1.80 1.10 Thr 228 . . B. . T . 0.00 −0.07 * . F 2.00 1.61 Asn 229 . . B . . T . 0.59 0.19 * . F1.20 1.52 Arg 230 . . B . . T . 0.12 −0.06 . . F 1.60 1.83 Thr 231 . . BB . . . 0.98 0.14 . * F 0.40 1.04 Ile 232 . . B B . . . 0.43 0.06 * * .0.05 1.04 Gln 233 . . B B . . . 0.44 0.30 * * . −0.30 0.40 Leu 234 . . BB . . . 0.20 0.69 * * . −0.60 0.37 Asn 235 . . B . . T . −0.12 0.96 * *. −0.20 0.82 Val 236 . . B . . T . −0.02 0.70 . * . −0.20 0.73 Ser 237 .. . . . T C 0.87 0.73 . * . 0.15 1.37 Tyr 238 . . . . . T C 0.87 0.44. * F 0.30 1.48 Pro 239 . . . . . . C 0.87 0.44 . * F 0.10 3.21 Pro 240. . . . T T . 0.56 0.49 . . F 0.50 1.97 Gln 241 . . . . T T . 0.56 0.59. . F 0.50 1.82 Asn 242 . . B . . T . 0.54 0.47 . . F −0.05 0.87 Leu 243. . B . . T . −0.07 0.53 . . . −0.20 0.81 Thr 244 . . B B . . . −0.560.74 * . . −0.60 0.35 Val 245 . . B B . . . −0.34 1.13 * . . −0.60 0.19Thr 246 . . B B . . . −0.69 1.13 . . . −0.60 0.39 Val 247 . . B B . . .−0.69 0.87 . * . −0.60 0.27 Phe 248 . . B B . . . −0.22 0.39 . . . −0.300.63 Gln 249 . . B B . . . −0.22 0.17 . . F 0.09 0.43 Gly 250 . . . . .T C 0.04 0.17 . . F 0.93 0.84 Glu 251 . . . . . T C 0.06 0.03 . * F 1.170.98 Gly 252 . . . . . T C 0.60 −0.37 . . F 2.01 0.76 Thr 253 . . . . .T C 0.71 −0.29 . * F 2.40 1.11 Ala 254 . . B . . . . −0.10 −0.21 . . F1.61 0.65 Ser 255 . . B . . . . −0.10 0.47 . . F 0.47 0.54 Thr 256 . . B. . . . −0.10 0.47 . . F 0.23 0.37 Ala 257 . . B . . . . −0.06 0.39 . .F 0.29 0.59 Leu 258 . . B . . . . −0.04 0.27 . . F 0.05 0.59 Gly 259 . .. . T . . 0.24 0.27 . . F 0.45 0.55 Asn 260 . . . . . T C −0.27 0.17 . .F 0.45 0.72 Ser 261 . . . . . T C −0.26 0.36 . . F 0.45 0.72 Ser 262 . .. . . T C −0.52 0.06 . . F 0.45 0.98 Ser 263 . . . . . T C −0.52 0.27 .. F 0.45 0.45 Leu 264 . A B . . . . −0.18 0.56 . . F −0.45 0.28 Ser 265. A B . . . . −0.52 0.17 . . . −0.30 0.36 Val 266 . A B . . . . −0.220.21 . . . −0.30 0.27 Leu 267 . A B . . . . −0.22 0.23 . . . −0.13 0.56Glu 268 . A B . . . . −0.73 −0.07 * . F 0.79 0.56 Gly 269 . . . . T T .0.19 0.23 * . F 1.16 0.62 Gln 270 . . . . T T . −0.32 −0.41 * . F 2.081.47 Ser 271 . . B . . T . −0.32 −0.41 * . F 1.70 0.70 Leu 272 . . B . .T . −0.18 0.23 * . F 0.93 0.53 Arg 273 . . B B . . . −0.77 0.37 * . .0.21 0.16 Leu 274 . . B B . . . −1.28 0.47 * * . −0.26 0.12 Val 275 . .B B . . . −1.28 0.73 * * . −0.43 0.11 Cys 276 . . B B . . . −1.280.04 * * . −0.30 0.09 Ala 277 . . B B . . . −0.47 0.43 * * . −0.60 0.15Val 278 . . B B . . . −0.79 0.14 * * . 0.00 0.33 Asp 279 . . . . T T .−0.19 −0.07 . . F 1.85 0.96 Ser 280 . . . . . T C 0.08 −0.21 . * F 2.101.46 Asn 281 . . . . . T C 0.86 −0.21 . * F 2.40 1.99 Pro 282 . . . . .T C 0.63 −0.86 . * F 3.00 2.34 Pro 283 . . . . T . . 1.19 −0.17 . * F2.40 1.44 Ala 284 . . . . T . . 0.90 −0.17 . * F 2.10 1.20 Arg 285 . . BB . . . 0.89 0.34 * * . 0.30 0.82 Leu 286 . . B B . . . 0.60 0.40 * * .−0.30 0.76 Ser 287 . . B B . . . 0.92 0.89 * * . −0.60 0.79 Trp 288 . .B B . . . 0.83 0.39 * * . −0.30 0.79 Thr 289 . . B B . . . 0.61 0.77 * *. −0.45 1.29 Trp 290 . . B B . . . 0.19 0.77 * * . −0.60 0.79 Arg 291 .. B B . . . 0.19 0.87 * * . −0.45 1.09 Ser 292 . . B B . . . 0.24 0.64 *. . −0.60 0.62 Leu 293 . . . B T . . 0.32 0.91 * . . −0.20 0.93 Thr 294. . . B T . . 0.33 0.43 * . . −0.20 0.73 Leu 295 . . . B . . C 0.620.81 * . . −0.40 0.73 Tyr 296 . . B . . T . 0.30 0.83 * . F 0.10 1.53Pro 297 . . . . T T . 0.30 0.57 . . F 0.62 1.64 Ser 298 . . . . T T .1.11 0.47 . . F 0.74 2.67 Gln 299 . . . . . T C 1.21 0.19 . . F 0.962.74 Pro 300 . . . . T T . 1.21 −0.14 . . F 1.88 2.74 Ser 301 . . . . .T C 0.60 0.11 . . F 1.20 1.69 Asn 302 . . . . . T C 0.00 0.37 . . F 0.930.72 Pro 303 . . B . . T . 0.30 0.66 * . F 0.31 0.39 Leu 304 . A B . . .. −0.51 0.23 * * F 0.09 0.50 Val 305 . A B . . . . −0.30 0.53 . * .−0.48 0.26 Leu 306 . A B . . . . −0.86 0.53 . * . −0.60 0.29 Glu 307 . AB . . . . −0.89 0.74 . * . −0.60 0.26 Leu 308 . A B . . . . −1.49 0.56. * . −0.60 0.47 Gln 309 . A B . . . . −1.02 0.60 * * . −0.60 0.47 Val310 . A B . . . . −0.17 0.34 * * . −0.30 0.27 His 311 . A B . . . . 0.640.34 * * . −0.30 0.55 Leu 312 . A . . . . C 0.30 −0.34 * * . 0.84 0.55Gly 313 . . . . . T C 1.11 −0.31 . * F 1.73 0.73 Asp 314 . . . . T T .0.41 −0.96 . * F 2.57 0.93 Glu 315 . . . . T T . 0.96 −0.67 . * F 2.910.97 Gly 316 . . . . T T . 0.32 −0.87 * * F 3.40 1.42 Glu 317 . A . . T. . 1.24 −0.73 * * F 2.51 0.46 Phe 318 . A B . . . . 1.00 −0.73 * * .1.62 0.52 Thr 319 . A B . . . . 1.00 −0.23 * * . 0.98 0.53 Cys 320 . A B. . . . 1.00 −0.26 * * . 0.89 0.53 Arg 321 . A . . T . . 1.04 0.14 * * .0.60 0.98 Ala 322 . A . . T . . 0.23 −0.26 * * F 1.60 0.91 Gln 323 . A .. T . . 0.59 −0.06 * * F 2.00 1.40 Asn 324 . . . . T T . 0.60 −0.20 * *F 2.50 0.71 Ser 325 . . . . . T C 1.27 0.19 * * F 1.45 0.94 Leu 326 . .. . . T C 1.12 0.09 * * F 1.20 0.94 Gly 327 . . . . T T . 0.86 0.19 . .F 1.15 0.79 Ser 328 . . B B . . . 0.56 0.43 . . F −0.20 0.44 Gln 329 . .B B . . . −0.26 0.43 . . F −0.45 0.71 His 330 . . B B . . . 0.04 0.43. * . −0.60 0.59 Val 331 . . B B . . . 0.04 0.40 . * . −0.60 0.71 Ser332 . A B . . . . 0.09 0.70 . * . −0.60 0.34 Leu 333 . A B . . . . −0.420.69 . * . −0.60 0.33 Asn 334 . A B . . . . −0.42 0.87 . * . −0.60 0.37Leu 335 . A . . . . C −0.39 0.63 . * . −0.40 0.48 Ser 336 . A . . . . C0.47 0.64 . * . −0.25 1.01 Leu 337 . A B . . . . 0.52 −0.04 . * . 0.451.09 Gln 338 . A B . . . . 1.02 0.31 . * F 0.00 2.06 Gln 339 . A B . . .. 0.68 0.11 . * F 0.34 2.22 Glu 340 . A B . . . . 1.53 0.16 . * F 0.682.66 Tyr 341 . . B . . T . 1.23 −0.53 * * F 2.32 3.08 Thr 342 . . . . TT . 2.16 −0.31 * * F 2.76 1.76 Gly 343 . . . . T T . 1.94 −0.71 * * F3.40 1.99 Lys 344 . . . . T T . 1.09 −0.29 * * F 2.76 1.96 Met 345 . . B. . . . 0.79 −0.40 . * F 1.82 1.01 Arg 346 . . B . . . . 0.69 −0.50 * *F 1.48 1.37 Pro 347 . . B . . T . 0.14 −0.50 * * F 1.19 0.68 Val 348 . .B . . T . −0.32 0.14 . * F 0.25 0.51 Ser 349 . . B . . T . −1.180.21 * * F 0.25 0.21 Gly 350 . . B . . T . −0.92 0.90 * . . −0.20 0.11Val 351 . . B B . . . −1.62 0.90 * . . −0.60 0.15 Leu 352 . . B B . . .−2.27 0.76 . . . −0.60 0.11 Leu 353 . . B B . . . −1.76 1.01 . . . −0.600.09 Gly 354 . . B B . . . −1.80 1.01 . . . −0.60 0.11 Ala 355 . . B B .. . −2.04 0.80 . . . −0.60 0.14 Val 356 . . B B . . . −1.53 0.61 . . .−0.60 0.17 Gly 357 . . B . . . . −1.31 0.36 . . . −0.10 0.17 Gly 358 . .. . . T C −0.81 0.43 . . F 0.15 0.17 Ala 359 . . B . . T . −1.06 0.41 .. F −0.05 0.33 Gly 360 . . B . . T . −1.28 0.27 . . . 0.10 0.33 Ala 361. . B . . T . −1.28 0.53 . . . −0.20 0.28 Thr 362 . . B B . . . −1.630.74 . . . −0.60 0.20 Ala 363 . . B B . . . −2.10 1.03 . . . −0.60 0.18Leu 364 . . B B . . . −1.81 1.29 . . . −0.60 0.15 Val 365 . . B B . . .−2.17 1.17 . . . −0.60 0.14 Phe 366 . . B B . . . −2.24 1.47 . . . −0.600.12 Leu 367 . . B B . . . −2.79 1.54 . . . −0.60 0.08 Ser 368 . . B B .. . −3.09 1.50 . . . −0.60 0.08 Phe 369 . . B B . . . −2.98 1.54 . . .−0.60 0.06 Cys 370 . . B B . . . −3.01 1.54 . . . −0.60 0.06 Val 371 . .B B . . . −3.17 1.54 . . . −0.60 0.03 Ile 372 . . B B . . . −3.211.80 * * . −0.60 0.03 Phe 373 . . B B . . . −2.80 1.66 * . . −0.60 0.04Ile 374 . . B B . . . −2.40 1.09 * . . −0.60 0.11 Val 375 . . B B . . .−2.40 0.83 * . . −0.26 0.20 Val 376 . . B B . . . −1.43 0.71 * . . 0.080.12 Arg 377 . . B . . T . −0.50 −0.07 * . . 1.72 0.35 Ser 378 . . . . TT . 0.24 −0.76 * . F 2.91 0.94 Cys 379 . . . . T T . 0.83 −1.40 * . F3.40 2.53 Arg 380 . . . . T T . 1.10 −1.66 * . F 3.06 1.73 Lys 381 . A .. T . . 2.07 −1.16 * . F 2.32 1.30 Lys 382 . A . . T . . 1.74 −1.54 * .F 1.98 4.76 Ser 383 . A . . . . C 1.46 −1.69 * . F 1.44 3.76 Ala 384 . AB . . . . 1.53 −1.19 * * F 0.90 1.90 Arg 385 . A B . . . . 1.42−0.69 * * F 0.75 0.96 Pro 386 . A B . . . . 0.52 −0.69 * * F 0.90 1.20Ala 387 . A B . . . . 0.13 −0.43 * . . 0.30 0.88 Ala 388 . A B . . . .0.43 −0.50 * * . 0.30 0.44 Asp 389 . . B . . T . 0.13 −0.50 * * . 0.700.48 Val 390 . . B . . T . −0.32 −0.24 * * . 0.70 0.33 Gly 391 . . B . .T . −0.71 −0.31 * . F 0.85 0.33 Asp 392 . . B . . T . −0.08 −0.20 * . F0.85 0.19 Ile 393 . A B . . . . 0.51 −0.20 * . F 0.45 0.52 Gly 394 . A B. . . . −0.08 −0.84 * . F 0.75 0.88 Met 395 . A B . . . . 0.78 −0.77 * .F 1.01 0.53 Lys 396 . A B . . . . 0.81 −0.37 * . F 1.12 1.22 Asp 397 . .B . . T . −0.08 −0.57 * * F 2.08 1.78 Ala 398 . . B . . T . 0.92−0.31 * * F 2.04 1.26 Asn 399 . . B . . T . 0.92 −0.93 * * F 2.60 1.23Thr 400 . . B . . T . 1.22 −0.50 * * F 1.89 0.73 Ile 401 . . B . . T .0.59 −0.11 * * F 1.63 0.97 Arg 402 . . B . . T . 0.29 −0.11 * . F 1.610.61 Gly 403 . . B . . T . 0.88 −0.13 * * F 1.59 0.57 Ser 404 . . . . .T C 0.53 −0.21 * * F 1.92 1.40 Ala 405 . . . . . . C 0.84 −0.47 * * F1.81 0.71 Ser 406 . . . . . T C 0.92 −0.07 * * F 2.40 1.15 Gln 407 . . .. . T C 0.50 0.19 * . F 1.41 0.71 Gly 408 . . . . . T C 0.84 0.29 . . F1.32 1.01 Asn 409 . . . . . T C 0.84 −0.21 . . F 1.68 1.30 Leu 410 . . .. . . C 1.14 −0.21 . . F 1.58 1.01 Thr 411 . . B . . T . 0.86 0.30 * . F1.08 1.07 Glu 412 . . B . . T . 0.86 0.37 * . F 1.27 0.67 Ser 413 . . B. . T . 1.20 −0.03 * . F 2.36 1.36 Trp 414 . . . . T T . 1.20 −0.71 * .F 3.40 1.58 Ala 415 . . . . . . C 1.80 −0.80 * * F 2.66 1.46 Asp 416 . .. . T . . 2.22 −0.37 * . F 2.56 1.69 Asp 417 . . . . . . C 2.19−0.76 * * F 2.66 3.15 Asn 418 . . . . . T C 2.46 −1.17 * * F 2.86 4.24Pro 419 . . . . . T C 2.40 −1.17 . . F 2.86 3.45 Arg 420 . . . . T T .2.18 −0.74 . * F 3.40 2.05 His 421 . . . . . T C 1.59 −0.06 . . . 2.411.05 His 422 . A . . . . C 1.00 0.04 . . . 0.92 0.69 Gly 423 . A B . . .. 0.97 0.11 . . . 0.38 0.35 Leu 424 . A B . . . . 0.88 0.61 . . . −0.260.35 Ala 425 . A B . . . . 0.47 0.50 . . . −0.30 0.35 Ala 426 . A . . .. C 0.16 0.39 . . . 0.50 0.47 His 427 . . . . . T C 0.19 0.39 . . . 1.200.57 Ser 428 . . . . . T C 0.53 −0.30 . * F 2.25 0.97 Ser 429 . . . . .T C 1.46 −0.80 . . F 3.00 1.66 Gly 430 . . . . . T C 2.04 −1.30 . . F2.70 2.39 Glu 431 . A . . . . C 1.74 −1.80 . . F 2.00 3.09 Glu 432 . A .. . . C 1.78 −1.50 * . F 1.70 1.62 Arg 433 . A B . . . . 1.83 −1.49 * .F 1.20 2.83 Glu 434 . A B . . . . 1.54 −1.16 * . F 0.90 2.56 Ile 435 . AB . . . . 1.68 −0.66 . . . 0.75 1.49 Gln 436 . A B . . . . 0.87 −0.23 *. . 0.45 1.18 Tyr 437 . A B . . . . 0.57 0.46 * . . −0.60 0.56 Ala 438 .. . . . . C −0.24 0.84 * . . −0.05 1.07 Pro 439 . . B . . . . −0.280.94 * . . −0.40 0.54 Leu 440 . . B . . . . 0.66 1.04 * . . −0.40 0.47Ser 441 . . . . . . C 0.31 0.29 * . . 0.10 0.92 Phe 442 . . . . . . C0.56 0.21 . . . 0.10 0.59 His 443 . . . . T . . 0.93 −0.21 . . . 1.051.24 Lys 444 . . . . T . . 1.14 −0.47 . . F 1.50 1.43 Gly 445 . . . . .. C 1.96 −0.46 . . F 1.60 2.86 Glu 446 . . . . . . C 1.44 −1.24 * . F2.20 3.51 Pro 447 . . . . . . C 1.84 −1.06 * . F 2.50 1.45 Gln 448 . . .. T . . 1.53 −0.67 . . F 3.00 1.96 Asp 449 . . . . . . C 1.49 −0.67 . .F 2.50 1.12 Leu 450 . . . . . T C 1.83 −0.27 . . F 2.10 1.26 Ser 451 . .. . . T C 1.24 −0.70 . . F 2.10 1.26 Gly 452 . . B . . T . 1.14 −0.60 .. F 1.45 0.76 Gln 453 . . . . . T C 1.14 −0.11 . . F 1.20 1.33 Glu 454 .. . . . . C 1.14 −0.40 . . F 1.00 1.60 Ala 455 . . . . . . C 1.96 −0.39. . F 1.30 2.59 Thr 456 . . . . . . C 2.01 −0.81 . . F 1.90 2.59 Asn 457. . . . . T C 2.06 −0.46 . . F 2.10 2.35 Asn 458 . . . . . T C 2.06−0.07 * . F 2.40 3.11 Glu 459 . . . . . T C 1.17 −0.57 * . F 3.00 3.73Tyr 460 . . . . T T . 1.80 −0.37 . * F 2.60 1.63 Ser 461 . . . . T . .1.22 −0.77 . * F 2.40 2.02 Glu 462 . . B . . . . 1.01 −0.49 . . F 1.250.82 Ile 463 . . B . . . . 1.06 −0.06 . . F 1.22 0.81 Lys 464 . . B . .. . 0.67 −0.81 . . F 1.64 1.21 Ile 465 . . B . . . . 0.52 −0.77 . * F1.76 0.89 Pro 466 . . B . . . . 0.43 −0.34 . * . 1.73 1.62 Lys 467 . . .. T . . 0.04 −0.60 . * . 2.70 1.04

TABLE VII Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . B. . . . 0.43 0.01 * . . −0.10 0.79 Asp 2 . . B . . . . 0.48 −0.41 * . .0.65 1.21 Leu 3 . . B . . T . 0.06 −0.41 * . . 0.70 0.94 Pro 4 A . . . .T . −0.41 −0.16 * . . 0.70 0.78 Arg 5 . . B . . T . −0.88 −0.13 * . F0.85 0.35 Gly 6 . . B . . T . −0.87 0.51 * . F −0.05 0.31 Leu 7 . . B B. . . −1.16 0.33 * . . −0.30 0.20 Val 8 . . B B . . . −0.93 0.81 * . .−0.60 0.11 Val 9 . . B B . . . −1.53 1.31 . . . −0.60 0.11 Ala 10 . . BB . . . −1.94 1.57 . * . −0.60 0.11 Trp 11 . . B B . . . −2.41 1.27 . .. −0.60 0.20 Ala 12 . . B B . . . −1.89 1.31 . . . −0.60 0.22 Leu 13 . .B B . . . −1.24 1.59 . . . −0.60 0.23 Ser 14 . . . . . . C −0.73 1.51 .. . −0.20 0.34 Leu 15 . . . . . . C −0.84 1.03 . . . −0.20 0.34 Trp 16 .. . . . T C −0.87 1.31 * . . 0.00 0.35 Pro 17 . . . . . T C −0.28 1.11 *. . 0.00 0.38 Gly 18 . . . . T T . 0.22 0.73 * . F 0.35 0.77 Phe 19 . .. . T T . −0.18 0.53 * . F 0.50 1.06 Thr 20 . . . . . . C 0.63 0.40 * .F 0.25 0.59 Asp 21 . . . . . . C 0.32 0.37 . * F 0.25 0.96 Thr 22 . . .. . . C 0.53 0.56 . * . 0.29 1.10 Phe 23 . . B . . . . 0.57 −0.23 * * .1.33 1.27 Asn 24 . . B . . T . 1.38 −0.23 * * . 1.87 1.10 Met 25 . . . .T T . 1.73 −0.23 * * . 2.61 1.49 Asp 26 . . . . T T . 1.52 −0.71 * . F3.40 3.44 Thr 27 . . . . T T . 1.94 −1.07 * * F 3.06 3.31 Arg 28 . . . .T . . 1.79 −1.47 * * F 2.52 6.55 Lys 29 . . B . . . . 0.90 −1.44 * . F1.78 2.91 Pro 30 . . B B . . . 1.29 −0.76 * . F 1.24 1.41 Arg 31 . . B B. . . 0.94 −0.81 . . F 0.90 1.12 Val 32 . . B B . . . 0.96 −0.39 . * .0.30 0.55 Ile 33 . . B . . T . 0.96 0.00 . * F 0.22 0.48 Pro 34 . . B .. T . 0.60 −0.43 . * F 0.79 0.48 Gly 35 . . . . T T . 0.22 0.06 * * F0.56 0.93 Ser 36 . . B . . T . −0.59 −0.09 * * F 0.88 1.34 Arg 37 . . BB . . . −0.43 0.01 . . F −0.30 0.75 Thr 38 . . B B . . . 0.11 0.37 . . F−0.27 0.66 Ala 39 . . B B . . . 0.08 0.37 . . . −0.39 0.49 Phe 40 . . BB . . . 0.11 0.74 . . . −0.66 0.39 Phe 41 . . B B . . . −0.44 1.23 . . .−0.63 0.39 Gly 42 . . B B . . . −0.56 1.39 . . . −0.60 0.29 Tyr 43 . . BB . . . −0.24 1.29 . . . −0.60 0.57 Thr 44 . . B B . . . 0.31 0.90 . . .−0.45 1.14 Val 45 . . B B . . . 1.01 0.61 . . . −0.45 1.57 Gln 46 . . BB . . . 0.82 0.19 . . . −0.15 1.67 Gln 47 . . B B . . . 0.87 0.11 . . .−0.30 0.81 His 48 . . B B . . . 0.77 0.01 . . . 0.10 1.47 Asp 49 . . B B. . . 1.08 −0.20 . . F 0.95 0.84 Ile 50 . . . B T . . 1.98 −0.20 . . F1.60 0.78 Ser 51 . . . . T T . 1.69 −0.60 . . F 2.70 1.14 Gly 52 . . . .T T . 0.88 −0.19 . . F 2.50 0.72 Asn 53 . . . . T T . 0.06 0.50 . . F1.35 0.85 Lys 54 . . . . T T . −0.80 0.46 . . F 1.10 0.47 Trp 55 . . B B. . . −0.26 0.71 . . . −0.10 0.35 Leu 56 . . B B . . . −0.54 0.71 . . .−0.35 0.22 Val 57 . . B B . . . −0.41 0.81 . . . −0.60 0.11 Val 58 . . BB . . . −1.22 1.24 . . . −0.60 0.16 Gly 59 . . B B . . . −1.27 1.01 . .. −0.60 0.16 Ala 60 . . B . . . . −1.29 0.33 . . . −0.10 0.38 Pro 61 . .. . . . C −0.48 0.17 . * . 0.10 0.73 Leu 62 . . . . . . C 0.03 −0.07 . .F 1.34 1.19 Glu 63 A . . . . T . 0.64 −0.07 . * F 1.68 1.16 Thr 64 . . B. . T . 0.99 0.19 . * F 1.42 1.18 Asn 65 . . . . T T . 1.62 0.16 . * F2.16 2.47 Gly 66 . . . . T T . 1.52 −0.53 . . F 3.40 2.86 Tyr 67 . . . .T . . 1.99 −0.04 . . F 2.56 2.86 Gln 68 . . . . T . . 1.99 −0.10 . . F2.48 1.76 Lys 69 . . B . T T . 1.44 −0.50 * . F 2.60 2.97 Thr 70 . . B .. T . 1.20 −0.29 * . F 2.12 1.41 Gly 71 . . B . . T . 1.59 −0.29 * . F2.04 1.27 Asp 72 . . B . . T . 1.17 −0.69 * . F 2.60 1.27 Val 73 . . B .. . . 0.96 −0.11 * . F 1.69 0.47 Tyr 74 . . B . . T . 0.06 −0.17 . . .1.48 0.74 Lys 75 . . B . . T . −0.52 0.04 . . . 0.62 0.33 Cys 76 . . B .. T . −0.21 0.73 . . . 0.06 0.31 Pro 77 . . B . . T . −0.56 0.59 . . .−0.20 0.27 Val 78 . . B . . . . 0.30 0.26 . . . −0.10 0.13 Ile 79 . . B. . . . −0.12 0.66 . . . −0.40 0.40 His 80 . . B . . T . −0.48 0.66 . .. −0.20 0.14 Gly 81 . . B . . T . 0.23 0.71 . . . −0.20 0.27 Asn 82 . .. . T T . −0.37 0.07 . . . 0.50 0.77 Cys 83 . . B . . T . 0.49 0.07 . *F 0.25 0.47 Thr 84 . . B . . . . 0.57 −0.03 . * F 0.65 0.76 Lys 85 . . B. . . . 0.26 0.23 . . F 0.05 0.39 Leu 86 . . B . . . . 0.71 0.26 . . .−0.10 0.72 Asn 87 . . B . . . . −0.14 −0.31 . . . 0.50 0.97 Leu 88 . . BB . . . 0.21 −0.16 . * . 0.30 0.36 Gly 89 . . B B . . . −0.29 0.33 . * .−0.30 0.63 Arg 90 . . B B . . . −0.63 0.33 . . . −0.30 0.32 Val 91 . . BB . . . 0.18 0.31 * . . −0.30 0.53 Thr 92 . . B B . . . −0.68 0.03 * . .−0.30 0.85 Leu 93 . . B B . . . −0.17 0.24 * * . −0.30 0.32 Ser 94 . . BB . . . 0.18 0.63 . * . −0.60 0.58 Asn 95 . . B B . . . 0.18 −0.01 . * F0.45 0.70 Val 96 . . B B . . . 1.08 −0.50 . . F 0.60 1.67 Ser 97 A . . .. . . 1.39 −1.19 * . F 1.10 2.49 Glu 98 A . . . . . . 2.20 −1.57 * . F1.10 2.58 Arg 99 A . . . . T . 1.90 −1.57 . * F 1.30 5.60 Lys 100 A . .. . T . 2.01 −1.60 . * F 1.30 4.13 Asp 101 A . . . . T . 2.06 −1.99 . *F 1.30 4.67 Asn 102 A . . . . T . 2.01 −1.30 . * . 1.15 1.97 Met 103 A .. . . . . 1.20 −0.87 . * . 0.80 0.97 Arg 104 A . . . . . . 0.79 −0.19. * . 0.50 0.48 Leu 105 . . B . . . . −0.07 0.20 * * . −0.10 0.40 Gly106 . . B . . . . −0.66 0.49 * * . −0.40 0.33 Leu 107 . . B . . . .−0.97 0.37 * * . −0.10 0.17 Ser 108 . . B . . . . −0.37 0.86 * * . −0.400.30 Leu 109 . . B . . . . −0.69 0.57 * * . −0.06 0.49 Ala 110 . . B . .. . 0.17 0.57 . * . 0.28 0.92 Thr 111 . . B . . . . 0.51 −0.11 . . F1.82 1.37 Asn 112 . . . . . T C 1.32 −0.50 . * F 2.56 2.78 Pro 113 . . .. T T . 1.32 −0.79 . . F 3.40 4.42 Lys 114 . . . . T T . 1.43 −0.90 . .F 3.06 4.10 Asp 115 . . . . T T . 1.21 −0.60 . . F 2.72 2.21 Asn 116 . .. . T T . 0.93 −0.31 . . F 2.08 1.18 Ser 117 . . B . . T . 0.27 −0.24 .. F 1.19 0.60 Phe 118 . . B . . T . 0.18 0.33 . . . 0.10 0.19 Leu 119 .. B . . T . −0.08 0.71 . . . −0.20 0.16 Ala 120 . . B . . . . −0.89 0.74. . . −0.40 0.18 Cys 121 . . B . . . . −1.18 1.04 . . . −0.40 0.17 Ser122 . . . . . T C −1.18 1.17 . . . 0.00 0.22 Pro 123 . . . . T T . −0.510.87 . . . 0.20 0.30 Leu 124 . . . . T T . 0.30 0.87 . . . 0.20 0.75 Trp125 . . . . T T . 0.22 0.30 . . . 0.50 0.97 Ser 126 . . B . . . . 0.540.49 . . . −0.27 0.34 His 127 . . B . . T . 0.54 0.49 . . . 0.06 0.40Glu 128 . . . . T T . 0.46 0.19 . . . 0.89 0.51 Cys 129 . . . . T T .1.02 −0.34 . . F 1.77 0.51 Gly 130 . . . . T T . 1.07 0.03 . . F 1.300.59 Ser 131 . . . . T T . 1.06 0.29 . . F 1.17 0.54 Ser 132 . . . . T T. 0.78 0.77 . . F 0.89 1.44 Tyr 133 . . . . T T . 0.43 0.69 . . F 0.762.11 Tyr 134 . . B . . T . 0.50 0.69 . . F 0.23 1.55 Thr 135 . . B . . .. 0.18 0.91 . . F −0.10 1.15 Thr 136 . . B . . . . 0.18 1.10 . . . −0.400.39 Gly 137 . . B . . T . 0.59 0.73 . * . −0.20 0.34 Met 138 . . B . .T . −0.02 −0.03 . * . 0.70 0.46 Cys 139 . . B . . T . 0.22 0.13 . * .0.10 0.23 Ser 140 . . B . . T . 0.23 0.04 . * . 0.10 0.38 Arg 141 . . B. . . . 0.54 0.00 * * F 0.05 0.52 Val 142 . . B . . . . 0.19 −0.21 * * F0.80 1.55 Asn 143 . . B . . T . 0.90 0.00 * * F 0.61 1.00 Ser 144 . . .. . T C 0.87 −0.39 * * F 1.62 1.00 Asn 145 . . . . T T . 0.87 0.40 * * F1.13 1.17 Phe 146 . . . . T T . 0.80 0.14 * * . 1.34 0.97 Arg 147 . . .. T . . 1.34 −0.26 * * . 2.10 1.45 Phe 148 . . . B T . . 0.49 −0.16 * *. 1.69 1.30 Ser 149 . . B B . . . 0.20 0.09 * * F 0.63 1.12 Lys 150 . .. B . . C −0.01 −0.20 * * F 1.07 0.58 Thr 151 . . . B T . . 0.100.23 * * F 0.61 1.03 Val 152 . . . B . . C −0.82 −0.06 * . . 0.50 0.77Ala 153 . . B B . . C −0.12 0.24 * . . −0.10 0.32 Pro 154 A A . . . . .0.29 0.64 * . . −0.60 0.38 Ala 155 A A . . . . . −0.42 0.16 * . . −0.151.01 Leu 156 A A . B . . . −0.11 0.09 * . . −0.30 0.54 Gln 157 A A . B .. . 0.43 −0.01 * . . 0.30 0.60 Arg 158 . A B B . . . 0.78 0.04 * . F−0.15 0.86 Cys 159 . A B B . . . 0.39 0.30 * * F 0.00 1.63 Gln 160 . . BB . . . 0.98 0.23 * * . −0.30 0.93 Thr 161 . . B B . . . 0.90 −0.17 * *. 0.30 0.80 Tyr 162 . . B B . . . 0.04 0.51 * * . −0.45 1.04 Met 163 . .B B . . . −0.96 0.59 . * . −0.60 0.45 Asp 164 . . B B . . . −1.14 0.87. * . −0.60 0.22 Ile 165 . . B B . . . −1.96 1.03 . * . −0.60 0.10 Val166 . . B B . . . −1.64 0.96 . * . −0.60 0.09 Ile 167 . . B B . . .−1.74 0.34 . * . −0.30 0.09 Val 168 . . B B . . . −1.44 0.77 . * . −0.350.12 Leu 169 . . B B . . . −1.44 0.47 . * . −0.10 0.22 Asp 170 . . . B T. . −0.86 0.23 * * F 1.00 0.50 Gly 171 . . . . T T . −0.89 −0.07 . . F2.25 0.90 Ser 172 . . . . T T . −0.24 −0.03 * * F 2.50 0.77 Asn 173 . .. . . T C 0.40 0.04 * . F 1.45 0.72 Ser 174 . . . . . T C 0.92 0.47 * .F 1.05 1.12 Ile 175 . . . B . . C 0.07 0.96 * . . 0.10 0.88 Tyr 176 . .. B . . C 0.41 1.21 * . . −0.15 0.41 Pro 177 . . B B . . . −0.14 0.81. * . −0.60 0.53 Trp 178 . . B B . . . −0.14 1.07 . * . −0.60 0.56 Val179 . . B B . . . 0.12 0.79 . . . −0.60 0.62 Glu 180 . . B B . . . 0.310.53 . * . −0.60 0.54 Val 181 A . . B . . . −0.26 0.89 . . . −0.60 0.45Gln 182 A . . B . . . −0.93 0.66 . . . −0.60 0.50 His 183 A . . B . . .−0.64 0.70 * * . −0.60 0.20 Phe 184 A . . B . . . −0.68 1.10 * . . −0.600.43 Leu 185 A . . B . . . −1.49 1.14 * . . −0.60 0.18 Ile 186 A . . B .. . −0.59 1.43 * . . −0.60 0.11 Asn 187 A . . B . . . −0.54 0.93 * . .−0.60 0.25 Ile 188 A . . B . . . −1.21 0.14 * * . −0.30 0.60 Leu 189 A .. B . . . −0.76 0.24 * * . −0.30 0.74 Lys 190 . . B B . . . −0.83 0.31 *. . −0.30 0.72 Lys 191 . . B B . . . −0.29 0.60 * * . −0.60 0.72 Phe 192. . B B . . . −0.50 0.34 * . . −0.30 0.86 Tyr 193 . . B B . . . 0.040.09 * . . −0.30 0.67 Ile 194 . . B B . . . 0.86 0.51 * . . −0.60 0.33Gly 195 . . . . . T C −0.08 0.91 * . . 0.00 0.66 Pro 196 . . . . T T .−0.12 0.81 . * F 0.35 0.30 Gly 197 . . . . T T . −0.28 0.46 . * F 0.350.73 Gln 198 . . B . . T . −0.38 0.41 . * F −0.05 0.55 Ile 199 . . B B .. . −0.34 0.41 . * . −0.60 0.35 Gln 200 . . B B . . . −0.86 0.63 . * .−0.60 0.26 Val 201 . . B B . . . −0.64 0.84 . * . −0.60 0.11 Gly 202 . .B B . . . −0.54 0.84 . * . −0.60 0.28 Val 203 . . B B . . . −0.89 0.91. * . −0.60 0.25 Val 204 . . B B . . . 0.00 0.94 . . . −0.60 0.34 Gln205 . . B B . . . 0.00 0.30 . . . −0.30 0.59 Tyr 206 . . B B . . . 0.00−0.13 * . . 0.45 1.32 Gly 207 . A B B . . . −0.51 −0.13 * . F 0.60 1.32Glu 208 A A . B . . . 0.31 −0.13 * . F 0.45 0.57 Asp 209 A A . B . . .1.17 −0.03 * . F 0.45 0.49 Val 210 A A . B . . . 0.47 −0.79 * . . 0.600.86 Val 211 A A . B . . . 0.68 −0.43 * . . 0.30 0.43 His 212 A A . B .. . 0.21 0.07 * * . −0.30 0.35 Glu 213 A A . . . . . 0.21 0.76 . . .−0.60 0.39 Phe 214 A A . . . . . 0.21 0.51 . * . −0.60 0.84 His 215 A A. . . . . 0.82 −0.13 * . . 0.45 1.04 Leu 216 A A . . . . . 1.79 0.13 * .. 0.04 0.94 Asn 217 A . . . . T . 1.52 0.13 * . . 0.93 2.12 Asp 218 A .. . . T . 0.67 −0.27 * . F 2.02 2.09 Tyr 219 . . . . T T . 1.41 −0.13 *. F 2.76 1.88 Arg 220 . . . . T T . 1.44 −0.81 * . F 3.40 2.34 Ser 221 A. . . . . . 1.40 −1.21 * . F 2.46 2.34 Val 222 . A B . . . . 0.54−0.57 * . F 1.92 1.11 Lys 223 . A B . . . . 0.54 −0.69 * . F 1.43 0.42Asp 224 . A B . . . . 0.20 −0.69 * . F 1.09 0.54 Val 225 A A . . . . .−0.50 −0.57 * . . 0.60 0.74 Val 226 A A . . . . . −0.50 −0.71 * . . 0.600.37 Glu 227 A A . . . . . 0.32 −0.33 * . . 0.30 0.30 Ala 228 A A . . .. . −0.61 0.17 * . . −0.30 0.55 Ala 229 A A . . . . . −0.61 0.21 * . .−0.30 0.52 Ser 230 A A . . . . . 0.24 −0.43 * * . 0.30 0.52 His 231 A A. . . . . 1.21 −0.03 * * . 0.64 0.89 Ile 232 A A . . . . . 0.87−0.53 * * . 1.43 1.72 Glu 233 A A . . . . . 1.11 −0.60 * * F 1.92 1.27Gln 234 . . . . T T . 1.39 −0.56 * * F 2.91 0.92 Arg 235 . . . . T T .1.69 −0.57 . * F 3.40 1.90 Gly 236 . . . . T T . 1.41 −1.26 * * F 3.061.90 Gly 237 . . . . . T C 2.41 −0.77 * * F 2.68 1.59 Thr 238 . . . . .. C 2.10 −1.17 * * F 2.30 1.59 Glu 239 . . . . . . C 1.51 −0.69 * * F2.12 2.31 Thr 240 . . B . . . . 0.70 −0.61 * * F 1.74 2.36 Arg 241 . . B. . . . 0.70 −0.26 . * F 1.60 1.42 Thr 242 . . B . . . . 0.16 −0.31 . *F 1.29 0.81 Ala 243 A A . . . . . 0.47 0.37 . * . 0.18 0.39 Phe 244 A A. . . . . −0.23 −0.11 . * . 0.62 0.35 Gly 245 A A . . . . . −0.51 0.67. * . −0.44 0.21 Ile 246 A A . . . . . −0.51 0.69 . * . −0.60 0.21 Glu247 A A . . . . . −0.50 0.19 . . . −0.30 0.47 Phe 248 A A . . . . . 0.09−0.21 . . . 0.30 0.64 Ala 249 A A . . . . . 0.20 −0.64 . . . 0.75 1.58Arg 250 A A . . . . . −0.16 −0.83 . . F 0.75 0.92 Ser 251 A A . . . . .0.73 −0.04 . . F 0.45 0.92 Glu 252 A A . . . . . 0.78 −0.43 . . F 0.601.58 Ala 253 A A . . . . . 1.13 −0.93 . . F 0.90 1.61 Phe 254 A A . . .. . 1.38 −0.50 * * F 0.60 1.19 Gln 255 A . . . . T . 1.38 −0.46 * . F0.85 0.68 Lys 256 A . . . . T . 1.72 −0.46 * . F 1.00 1.32 Gly 257 A . .. . T . 1.38 −0.96 * . F 1.30 3.05 Gly 258 A . . . . T . 1.38 −1.31 * .F 1.30 1.74 Arg 259 A A . . . . . 2.12 −1.21 * . F 0.75 0.88 Lys 260 A A. . . . . 2.17 −1.21 * . F 0.90 1.78 Gly 261 A A . . . . . 1.27 −1.64 *. F 0.90 3.59 Ala 262 A A . . . . . 1.01 −1.43 * . F 0.90 1.36 Lys 263 A. . B . . . 0.47 −0.81 * . F 0.75 0.67 Lys 264 . . B B . . . −0.50−0.13 * . . 0.30 0.48 Val 265 . . B B . . . −1.43 0.09 * . . −0.30 0.35Met 266 . . B B . . . −1.40 0.27 * . . −0.30 0.12 Ile 267 . . B B . . .−0.81 0.76 * . . −0.60 0.09 Val 268 . . B B . . . −1.20 0.76 * . . −0.600.20 Ile 269 . . B . . T . −1.24 0.54 . . . −0.20 0.20 Thr 270 . . B . .T . −0.69 −0.07 . . F 0.85 0.49 Asp 271 . . B . . T . −0.12 −0.37 . . F0.85 0.89 Gly 272 . . . . . T C 0.77 −0.51 . . F 1.50 1.73 Glu 273 . . .. . . C 1.32 −1.20 . . F 1.60 2.00 Ser 274 . . . . . . C 2.00 −1.30 . .F 1.90 1.60 His 275 . . . . . . C 2.31 −0.87 . . F 2.20 2.51 Asp 276 . .. . . . C 1.50 −1.30 . * F 2.50 2.42 Ser 277 . . . . . T C 1.84 −0.61 .. F 3.00 1.49 Pro 278 A . . . . T . 1.89 −1.00 . . F 2.50 1.89 Asp 279 A. . . . T . 1.33 −1.50 * . F 2.20 2.27 Leu 280 A . . . . T . 0.48−0.86 * . F 1.90 1.25 Glu 281 A A . . . . . 0.48 −0.56 * . F 1.05 0.57Lys 282 A A . . . . . 0.78 −0.59 * . F 0.75 0.59 Val 283 A A . . . . .0.69 −0.19 * . . 0.45 1.24 Ile 284 A A . . . . . 0.69 −0.49 * . F 0.450.96 Gln 285 . A B . . . . 1.61 −0.49 * . F 0.79 0.83 Gln 286 A A . . .. . 1.61 −0.49 * . F 1.28 2.19 Ser 287 A A . . . . . 1.57 −1.13 * * F1.92 5.22 Glu 288 . . . . . T C 1.57 −1.41 * . F 2.86 4.85 Arg 289 . . .. T T . 2.14 −1.17 * . F 3.40 2.08 Asp 290 . . . . T T . 2.26 −1.09 . .F 3.06 2.24 Asn 291 . . . . T T . 2.01 −1.47 * . F 2.72 2.53 Val 292 . .B B . . . 1.72 −0.71 . . F 1.58 2.02 Thr 293 . . B B . . . 0.87 −0.21 .. F 0.94 1.22 Arg 294 . . B B . . . 0.17 0.43 * . . −0.60 0.57 Tyr 295 .. B B . . . −0.69 0.53 * . . −0.60 0.77 Ala 296 . . B B . . . −1.500.53 * . . −0.60 0.40 Val 297 . . B B . . . −0.99 0.73 * . . −0.60 0.17Ala 298 . . B B . . . −0.92 1.16 * . . −0.60 0.11 Val 299 . . B B . . .−1.28 1.16 * . . −0.60 0.16 Leu 300 . . B B . . . −1.03 1.41 * . . −0.600.34 Gly 301 . . B B . . . −0.33 1.17 * . . −0.60 0.55 Tyr 302 . . B B .. . 0.63 0.67 * . . −0.45 1.45 Tyr 303 . . B . . . . 0.88 0.03 * . .0.39 3.44 Asn 304 . . B . . T . 0.84 −0.23 * . . 1.53 3.44 Arg 305 . . .. T T . 1.66 0.03 * . F 1.82 1.54 Arg 306 . . . . T T . 1.79 −0.33 * . F2.76 1.58 Gly 307 . . . . T T . 2.03 −0.66 * . F 3.40 1.52 Ile 308 . . .. . . C 1.97 −1.06 . . F 2.66 1.34 Asn 309 . . . . . T C 1.27 −0.57 * .F 2.37 0.99 Pro 310 . . . . . T C 0.34 0.21 * . F 1.13 0.86 Glu 311 . .B . . T . 0.23 0.47 . . F 0.44 1.02 Thr 312 . . B . . T . 0.58 0.19 * .F 0.40 1.02 Phe 313 A A . . . . . 0.58 −0.21 . * . 0.45 1.14 Leu 314 A A. . . . . 0.62 0.04 * * . −0.30 0.46 Asn 315 A A . . . . . 0.59 0.04 * .. −0.30 0.64 Glu 316 A A . . . . . −0.30 0.31 * . F 0.00 1.16 Ile 317 AA . . . . . −0.58 0.21 * . . −0.30 0.98 Lys 318 A A . . . . . −0.180.03 * . . −0.30 0.62 Tyr 319 . A B . . . . 0.63 0.01 * . . −0.30 0.48Ile 320 . A B . . . . 0.42 0.01 * . . −0.15 1.14 Ala 321 . A B . . . .0.42 −0.24 * . . 0.64 0.88 Ser 322 . A B . . . . 1.31 −0.24 * . . 0.980.94 Asp 323 . . . . . T C 1.31 −1.00 . . F 2.52 2.23 Pro 324 A . . . .T . 1.52 −1.69 . . F 2.66 4.42 Asp 325 . . . . T T . 1.71 −1.69 . . F3.40 4.49 Asp 326 A . . . . T . 1.60 −1.29 . . F 2.66 2.33 Lys 327 A A .. . . . 1.90 −0.50 . . F 1.62 1.30 His 328 A A . . . . . 1.04 −0.53 . *. 1.43 1.26 Phe 329 . A B . . . . 0.94 0.11 * . . 0.04 0.56 Phe 330 . AB . . . . 0.94 0.60 * . . −0.60 0.40 Asn 331 A A . . . . . 0.94 0.60 * .. −0.60 0.49 Val 332 A A . . . . . 0.31 0.10 * . . −0.30 0.99 Thr 333 AA . . . . . −0.24 −0.19 * . F 0.60 1.15 Asp 334 A A . . . . . −0.36−0.47 * * F 0.45 0.72 Glu 335 A A . . . . . 0.39 −0.19 . * . 0.30 0.81Ala 336 A A . . . . . 0.39 −0.83 . . . 0.75 1.12 Ala 337 A A . . . . .0.36 −1.31 . . . 0.75 1.12 Leu 338 A A . . . . . −0.19 −0.63 * . . 0.600.45 Lys 339 A A . . . . . −0.19 0.01 * . . −0.30 0.33 Asp 340 A A . . .. . −0.78 −0.49 * . . 0.30 0.55 Ile 341 A A . . . . . −1.00 −0.49 * . .0.30 0.67 Val 342 A A . . . . . −0.76 −0.49 * . . 0.30 0.28 Asp 343 A A. . . . . 0.06 −0.06 * . . 0.30 0.16 Ala 344 A A . . . . . 0.12 −0.06 *. . 0.30 0.39 Leu 345 A . . . . T . −0.77 −0.74 * . . 1.15 1.03 Gly 346A . . . . T . −0.58 −0.70 * . . 1.00 0.43 Asp 347 A . . . . T . −0.020.09 * . . 0.10 0.37 Arg 348 . . B . . T . −0.83 −0.03 * . . 0.70 0.60Ile 349 . . B . . . . −0.24 −0.03 * . . 0.50 0.50 Phe 350 . . B . . . .0.22 −0.46 * . . 0.50 0.52 Ser 351 . . B . . . . 0.26 −0.03 * . . 0.500.26 Leu 352 . . B . . . . 0.26 0.46 . . . −0.10 0.54 Glu 353 . . . . .. C 0.19 0.17 . . F 1.00 1.01 Gly 354 . . . . . . C 1.08 −0.61 . . F2.20 1.50 Thr 355 . . . . . . C 1.78 −0.60 . * F 2.50 2.93 Asn 356 . . .. . T C 1.77 −1.29 . . F 3.00 2.93 Lys 357 . . . . . T C 2.28 −0.80 . *F 2.70 4.28 Asn 358 . . . . . T C 1.58 −0.84 . . F 2.40 3.97 Glu 359 A .. . . T . 1.58 −0.54 . . F 1.90 2.14 Thr 360 A . . . . T . 1.08 −0.51. * F 1.60 1.06 Ser 361 A . . . . T . 1.08 0.17 . * F 0.25 0.54 Phe 362A . . . . T . 0.43 −0.23 . * . 0.70 0.54 Gly 363 A . . . . T . 0.13 0.39. . . 0.10 0.37 Leu 364 A . . . . . . 0.13 0.29 . * . −0.10 0.37 Glu 365A . . . . . . 0.13 0.30 . . . −0.10 0.74 Met 366 A . . . . . . 0.09 0.00. . . 0.05 1.09 Ser 367 A . . . . T . 0.09 0.00 . . F 0.40 1.30 Gln 368A . . . . T . 0.13 0.10 . . F 0.25 0.65 Thr 369 . . . . . T C 0.64 0.49. . F 0.15 0.88 Gly 370 . . . . . T C 0.61 0.26 . . F 0.45 0.88 Phe 371. . . . . . C 0.36 0.37 . . F 0.25 0.69 Ser 372 . . . B . . C −0.20 0.61. . F −0.25 0.36 Ser 373 . . . B . . C −0.20 0.77 . . . −0.40 0.27 His374 . . B B . . . 0.11 0.34 . . . −0.30 0.53 Val 375 . . B B . . . 0.11−0.44 . . . 0.30 0.67 Val 376 . . B . . T . −0.04 −0.40 . . . 0.70 0.49Glu 377 . . B . . T . −0.56 −0.14 . . . 0.70 0.27 Asp 378 A . . . . T .−1.07 0.04 . . . 0.10 0.30 Gly 379 A . . . . T . −1.38 0.09 . . . 0.100.33 Val 380 A A . . . . . −1.11 −0.13 . . . 0.30 0.19 Leu 381 A A . . .. . −1.11 0.37 . . . −0.30 0.11 Leu 382 . A B . . . . −1.46 1.01 . . .−0.60 0.09 Gly 383 . A B . . . . −2.04 1.01 . . . −0.60 0.11 Ala 384 . AB . . . . −1.94 0.87 . . . −0.60 0.14 Val 385 . A B . . . . −1.09 0.94 .. . −0.60 0.27 Gly 386 . . B . . . . −0.57 0.26 . . . −0.10 0.45 Ala 387. . B . . . . 0.24 0.74 . . . −0.40 0.47 Tyr 388 . . B . . . . 0.24 0.64. . . −0.25 1.01 Asp 389 . . . . T . . 0.24 0.43 . . . 0.15 1.01 Trp 390A . . . . . . 0.24 0.50 . . . −0.25 1.01 Asn 391 A . . . . . . −0.220.64 * . . −0.40 0.48 Gly 392 A A . . . . . 0.41 0.57 * . . −0.60 0.24Ala 393 A A . . . . . 0.66 0.57 . . . −0.60 0.45 Val 394 A A . . . . .0.34 −0.34 . . . 0.30 0.49 Leu 395 A A . . . . . 0.33 −0.26 . . F 0.450.71 Lys 396 A A . . . . . −0.26 −0.30 . . F 0.45 0.94 Glu 397 A A . . .. . −0.26 −0.30 . . F 0.60 1.28 Thr 398 A A . . . . . 0.38 −0.51 . . F0.90 1.54 Ser 399 A . . . . T . 0.38 −1.20 . . F 1.30 1.54 Ala 400 A . .. . T . 0.30 −0.56 . . F 1.15 0.66 Gly 401 A . . . . T . 0.04 0.13 . . F0.25 0.32 Lys 402 . . B . . T . −0.77 0.07 . . F 0.25 0.37 Val 403 . . B. . . . −0.34 0.37 . . . −0.10 0.30 Ile 404 . . B . . . . −0.04 −0.13 .. . 0.50 0.60 Pro 405 . . B . . . . 0.24 −0.56 . . . 0.80 0.52 Leu 406 .. B . . . . 0.34 −0.17 . . . 0.50 0.93 Arg 407 A . . . . . . −0.51 −0.06. . F 0.80 2.08 Glu 408 A . . . . . . 0.39 −0.06 . . F 0.80 1.11 Ser 409A . . . . . . 1.28 −0.49 . * F 0.80 2.69 Tyr 410 A A . . . . . 0.79−1.17 . . F 0.90 2.38 Leu 411 A A . . . . . 1.39 −0.39 . . F 0.60 1.19Lys 412 A A . . . . . 1.28 0.04 . . F 0.00 1.37 Glu 413 A A . . . . .1.28 −0.34 * . F 0.60 1.52 Phe 414 A A . . . . . 0.77 −1.10 * . F 0.903.19 Pro 415 A A . . . . . 1.06 −1.10 * . F 0.90 1.31 Glu 416 A A . . .. . 1.87 −1.10 * . F 0.90 1.52 Glu 417 A A . . . . . 1.79 −0.70 * * F0.90 2.82 Leu 418 A A . . . . . 1.44 −0.99 * * F 0.90 2.48 Lys 419 A A .. . . . 1.56 −0.99 * * F 0.90 1.42 Asn 420 A . . . . T . 1.52 −0.49 * .. 0.70 0.83 His 421 A . . . . T . 0.71 0.27 * . . 0.25 1.57 Gly 422 A .. . . T . 0.37 0.27 . . . 0.10 0.65 Ala 423 . . B . . T . 0.93 0.70 . .. −0.20 0.40 Tyr 424 . . B B . . . 0.58 1.06 . . . −0.60 0.46 Leu 425 .. B B . . . −0.28 1.04 . . . −0.60 0.67 Gly 426 . . B B . . . −0.56 1.26. . . −0.60 0.49 Tyr 427 . . B B . . . −0.51 1.24 . . . −0.60 0.45 Thr428 . . B B . . . −0.78 0.87 . . . −0.60 0.74 Val 429 . . B B . . .−1.39 0.83 * . . −0.60 0.55 Thr 430 . . B B . . . −0.88 1.04 * . . −0.600.26 Ser 431 . . B B . . . −0.83 0.67 . . . −0.60 0.24 Val 432 . . B B .. . −0.48 0.57 . . . −0.60 0.44 Val 433 . . B B . . . −0.17 −0.07 . . F0.79 0.60 Ser 434 . . B . . T . 0.34 −0.16 . . F 1.53 0.77 Ser 435 . . B. . T . 0.77 −0.11 . . F 2.02 1.03 Arg 436 . . B . . T . 0.21 −0.76 . .F 2.66 2.71 Gln 437 . . . . T T . 0.82 −0.76 . * F 3.40 1.50 Gly 438 . .B B . . . 0.82 −0.39 * * F 1.96 1.75 Arg 439 . . B B . . . 0.53−0.13 * * F 1.47 0.66 Val 440 . . B B . . . 0.49 0.37 * * . 0.38 0.39Tyr 441 . . B B . . . −0.21 0.40 * * . −0.26 0.39 Val 442 . . B B . . .−0.42 0.47 * * . −0.60 0.20 Ala 443 . . B B . . . 0.03 0.90 * * . −0.600.42 Gly 444 . . B B . . . −0.78 0.26 * * . −0.30 0.52 Ala 445 . . B . .. . 0.08 0.29 . * . −0.10 0.61 Pro 446 . . . . . . C 0.29 0.04 * * F0.25 0.97 Arg 447 . . B . . . . 0.83 0.04 * * F 0.20 1.33 Phe 448 . . B. . . . 1.08 0.10 * * . 0.18 1.90 Asn 449 . . . . T . . 1.47 0.03 * * .0.71 1.22 His 450 . . . . T T . 1.20 −0.40 * * F 1.79 1.24 Thr 451 . . .. . T C 0.52 0.24 * * F 1.12 1.06 Gly 452 . . . . T T . −0.40 0.14 * * F1.30 0.46 Lys 453 . . B . . T . −0.40 0.43 * * F 0.47 0.28 Val 454 . . BB . . . −0.71 0.71 . . . −0.21 0.17 Ile 455 . . B B . . . −1.28 0.71 . .. −0.34 0.25 Leu 456 . . B B . . . −1.00 0.90 . . . −0.47 0.12 Phe 457 .. B B . . . −0.66 1.40 . . . −0.60 0.22 Thr 458 . . B B . . . −0.70 1.16. . . −0.60 0.51 Met 459 . . B B . . . 0.27 0.87 . . . −0.32 1.00 His460 . . . . . T C 0.86 0.19 . . . 1.01 2.26 Asn 461 . . . . . T C 0.86−0.21 . . F 2.04 2.10 Asn 462 . . . . . T C 1.24 −0.01 . . F 2.32 1.75Arg 463 . . . . T T . 0.67 −0.14 . . F 2.80 1.85 Ser 464 . . . . . . C1.23 0.04 . . F 1.37 0.81 Leu 465 A A . . . . . 1.27 0.14 . . . 0.540.68 Thr 466 . A B . . . . 0.68 0.14 . . . 0.26 0.60 Ile 467 . A B . . .. 0.08 0.64 . * . −0.32 0.46 His 468 . A B . . . . 0.08 0.87 . * . −0.600.55 Gln 469 . A B . . . . 0.03 0.19 * * . −0.30 0.74 Ala 470 . A B . .. . 0.84 0.13 * * . 0.02 1.05 Met 471 . . B . . T . 1.16 −0.16 * . .1.19 1.33 Arg 472 . . B . . T . 1.16 −0.26 * * F 1.51 1.33 Gly 473 . . B. . T . 0.84 0.03 * . F 0.93 0.92 Gln 474 . . B . . T . 0.54 −0.04 . . F1.70 0.92 Gln 475 . . B . . . . 0.89 −0.27 * . F 1.33 0.63 Ile 476 . . B. . . . 0.79 0.49 * . F 0.41 1.00 Gly 477 . . B . . T . 0.33 0.84 * . F0.29 0.50 Ser 478 . . B . . T . 0.38 0.87 * . F 0.12 0.29 Tyr 479 . . .. . T C 0.38 0.86 * . . 0.00 0.55 Phe 480 . . B . . T . −0.51 0.17 * . F0.25 0.96 Gly 481 . . . B . . C 0.07 0.43 * . F −0.25 0.50 Ser 482 . . .B . . C 0.11 0.53 * . F −0.25 0.46 Glu 483 . . B B . . . −0.44 0.16 * .F −0.15 0.71 Ile 484 . . B B . . . −0.20 0.01 . . F −0.15 0.54 Thr 485 .. B B . . . −0.39 −0.41 . * F 0.45 0.67 Ser 486 . . B B . . . −0.04−0.11 . * F 0.45 0.27 Val 487 . . B B . . . −0.09 −0.11 . * F 0.76 0.64Asp 488 . . B B . . . −0.09 −0.37 . * F 1.07 0.44 Ile 489 . . B . . . .0.46 −0.86 . * F 1.88 0.55 Asp 490 . . . . T T . −0.09 −0.81 . * F 2.790.73 Gly 491 . . . . T T . −0.10 −0.81 . * F 3.10 0.33 Asp 492 . . . . TT . 0.76 −0.33 * * F 2.49 0.67 Gly 493 . . B . . T . −0.10 −1.01 * * F2.08 0.67 Val 494 . . B B . . . −0.02 −0.37 . * F 1.07 0.50 Thr 495 . .B B . . . −0.83 −0.11 . . F 0.76 0.25 Asp 496 . . B B . . . −1.34 0.57 *. F −0.45 0.21 Val 497 . . B B . . . −1.69 0.79 * . . −0.60 0.21 Leu 498. . B B . . . −1.93 0.57 . . . −0.60 0.14 Leu 499 . . B B . . . −1.290.59 . . . −0.60 0.09 Val 500 . . B B . . . −1.58 1.01 . . . −0.60 0.18Gly 501 . . B B . . . −1.82 0.99 . . . −0.60 0.21 Ala 502 . . B . . . .−1.67 1.06 . . . −0.40 0.41 Pro 503 . . B . . . . −0.86 1.16 . . . −0.400.48 Met 504 . . B . . . . −0.04 0.91 . . . −0.40 0.77 Tyr 505 . . B . .. . 0.47 0.49 . . . −0.25 1.33 Phe 506 . . B . . . . 0.92 0.41 * . .−0.40 0.85 Asn 507 A . . . . T . 1.51 −0.01 . * . 0.85 1.68 Glu 508 A .. . . T . 1.83 −0.63 . * F 1.30 1.86 Gly 509 A . . . . T . 2.09 −1.39. * F 1.30 4.20 Arg 510 A . . . . T . 2.38 −1.74 . * F 1.30 2.58 Glu 511A . . . . . . 2.22 −2.14 . * F 1.10 2.98 Arg 512 . . . B T . . 1.98−1.50 . * F 1.30 2.24 Gly 513 . . . B T . . 1.12 −1.17 . * F 1.30 1.79Lys 514 . . B B . . . 1.22 −0.53 . * F 0.75 0.77 Val 515 . . B B . . .1.11 0.23 . * . −0.30 0.61 Tyr 516 . . B B . . . 0.30 0.23 . * . −0.151.07 Val 517 . . B B . . . 0.30 0.49 . * . −0.60 0.44 Tyr 518 . . B . .. . 0.64 0.49 * * . 0.01 1.17 Glu 519 . . B . . . . 0.60 0.24 * . . 0.571.29 Leu 520 . . B . . . . 1.57 −0.11 . . . 1.43 2.80 Arg 521 A . . . .T . 1.11 −0.76 . . F 2.34 3.50 Gln 522 . . B . . T . 1.11 −0.73 . . F2.60 1.75 Asn 523 . . B . . T . 1.11 −0.09 * . F 2.04 1.57 Arg 524 . . B. . T . 1.11 −0.01 * * F 1.78 1.26 Phe 525 . . B . . . . 1.58 0.39 * . .0.57 1.17 Val 526 . . B . . T . 1.16 0.41 * * . 0.06 0.72 Tyr 527 . . B. . T . 0.34 0.50 . * . −0.20 0.53 Asn 528 . . B . . T . 0.39 1.19 . * F0.29 0.50 Gly 529 . . . . T T . 0.28 0.40 * * F 1.18 1.36 Thr 530 . . .. . . C 0.68 −0.24 . . F 2.02 1.45 Leu 531 . . . . . T C 1.50 −0.61 . .F 2.86 1.21 Lys 532 . . . . T T . 1.44 −0.51 . * F 3.40 1.66 Asp 533 . .B . . T . 1.20 −0.56 . * F 2.66 1.54 Ser 534 . . B . . T . 1.54 −0.29 .. F 2.02 2.93 His 535 . . B . . T . 1.86 −0.57 . . . 1.83 2.54 Ser 536 .. B . . T . 2.08 −0.17 . * . 1.19 2.44 Tyr 537 . . B . . T . 2.14 0.33. * . 0.25 1.84 Gln 538 . . B . . T . 1.44 −0.06 . * . 0.85 2.65 Asn 539. . B . . . . 1.40 0.23 . * . 0.05 1.71 Ala 540 . . B . . . . 1.13 0.27. * . 0.05 1.08 Arg 541 . . B . . . . 1.13 −0.10 * * . 0.50 0.84 Phe 542. . B . . . . 0.49 −0.11 * * F 0.65 0.70 Gly 543 . . . . T T . −0.100.17 * * F 0.65 0.48 Ser 544 . . . . . T C −0.40 0.17 * * F 0.45 0.25Ser 545 . . B . . T . −0.67 0.56 . * F −0.05 0.39 Ile 546 . . B . . T .−0.67 0.41 * * . −0.20 0.29 Ala 547 . . B B . . . 0.03 −0.01 * . . 0.300.42 Ser 548 . . B B . . . −0.43 −0.40 . . . 0.30 0.53 Val 549 . . B B .. . −0.13 −0.10 * . . 0.64 0.62 Arg 550 . . B B . . . 0.17 −0.39 * . F1.13 0.99 Asp 551 . . B . . . . 1.06 −0.49 * . F 1.82 1.28 Leu 552 . . B. . . . 1.34 −0.87 * . F 2.46 2.88 Asn 553 . . . . T T . 1.40 −1.13 * .F 3.40 1.97 Gln 554 . . . . T T . 2.26 −0.37 * . F 2.76 1.85 Asp 555 . .. . T T . 2.14 0.03 * . F 1.82 3.60 Ser 556 . . . . T T . 1.29 −0.66 * .F 2.38 3.74 Tyr 557 . . . B T . . 1.24 −0.41 . . F 1.34 1.60 Asn 558 . .B B . . . 0.39 −0.17 . . F 0.45 0.71 Asp 559 . . B B . . . 0.04 0.47 . .. −0.60 0.39 Val 560 . . B B . . . −0.54 0.51 * . . −0.60 0.25 Val 561 .. B B . . . −0.46 0.26 . . . −0.30 0.16 Val 562 . . B B . . . −1.02 0.29. . . −0.30 0.14 Gly 563 . . B B . . . −1.02 0.97 . . . −0.60 0.16 Ala564 . A B . . . . −1.02 0.33 . . . −0.30 0.38 Pro 565 A A . . . . .−0.17 −0.31 . . . 0.30 0.85 Leu 566 A A . . . . . 0.66 −0.56 . . . 0.751.37 Glu 567 A A . . . . . 0.92 −0.49 . . F 0.60 1.85 Asp 568 A A . . .. . 0.92 −0.49 . . . 0.45 1.21 Asn 569 A A . . . . . 0.92 −0.49 . . .0.45 1.45 His 570 A A . . . . . 0.24 −0.67 . . . 0.60 0.85 Ala 571 A . .B . . . 0.81 0.01 . . . −0.30 0.36 Gly 572 A . . B . . . −0.08 0.77 . .. −0.60 0.35 Ala 573 A . . B . . . −0.78 1.06 . . . −0.60 0.18 Ile 574 .. B B . . . −0.81 1.34 . . . −0.60 0.15 Tyr 575 . . B B . . . −1.12 1.34. . . −0.60 0.21 Ile 576 . . B B . . . −1.23 1.34 * * . −0.60 0.21 Phe577 . . B B . . . −0.78 1.63 * * . −0.60 0.25 His 578 . . B B . . .−0.53 0.94 * * . −0.60 0.32 Gly 579 . . . B T . . 0.06 0.61 * * . −0.200.45 Phe 580 . . . . T T . −0.59 0.31 * * . 0.50 0.69 Arg 581 . . . . TT . −0.51 0.21 * * F 0.65 0.36 Gly 582 . . . . T T . 0.23 0.40 . . F0.35 0.30 Ser 583 . . . . T T . −0.04 −0.03 . . F 1.25 0.69 Ile 584 . .. . . . C 0.09 −0.33 * * F 1.15 0.51 Leu 585 . . . . . . C 0.83 0.10 * *F 0.85 0.79 Lys 586 . . . . . . C 0.72 −0.33 . * F 1.90 1.18 Thr 587 . .. . . T C 1.18 −0.31 * * F 2.40 2.92 Pro 588 . . . . . T C 0.59−1.00 * * F 3.00 6.94 Lys 589 . . B . . T . 1.17 −1.00 * * F 2.50 2.43Gln 590 . . B . . T . 1.39 −0.51 * * F 2.20 2.43 Arg 591 . . B B . . .1.04 −0.50 . * F 1.20 1.59 Ile 592 . . B B . . . 1.36 −0.54 . * F 1.201.06 Thr 593 . A B B . . . 0.76 −0.54 . * F 0.90 1.06 Ala 594 . A B B .. . 0.12 −0.26 . * F 0.45 0.45 Ser 595 . A B . . . . −0.19 0.24 . . F−0.15 0.65 Glu 596 . A B . . . . −0.64 0.04 . * F −0.15 0.65 Leu 597 A A. B . . . −0.57 −0.01 . . F 0.45 0.63 Ala 598 A A . B . . . −0.26 0.17 *. . −0.30 0.39 Thr 599 A A . B . . . 0.09 0.19 * . . −0.30 0.39 Gly 600A A . B . . . −0.31 0.94 * . . −0.60 0.74 Leu 601 . . B B . . . −0.661.04 * . . −0.60 0.63 Gln 602 . . B B . . . −0.51 0.97 * . . −0.60 0.44Tyr 603 . . . . T T . −0.22 1.06 * . . 0.20 0.24 Phe 604 . . B . . T .−0.80 1.01 * . . −0.20 0.38 Gly 605 . . B . . T . −0.49 1.01 * . . −0.200.15 Cys 606 . . B . . T . −0.02 1.11 . * . −0.20 0.13 Ser 607 . . B B .. . −0.02 0.79 . * . −0.60 0.15 Ile 608 . . B B . . . −0.59 0.40 . * .−0.60 0.27 His 609 . . B B . . . 0.11 0.66 . * . −0.60 0.41 Gly 610 . .B B . . . −0.36 0.09 . * . −0.30 0.52 Gln 611 . . B . . . . 0.31 0.39. * . −0.10 0.61 Leu 612 . . B . . . . 0.61 0.10 . * . −0.10 0.72 Asp613 . . B . . . . 1.50 −0.40 . * . 0.65 1.26 Leu 614 . . B . . . . 1.19−0.83 . * . 0.95 1.21 Asn 615 A . . . . T . 0.72 −0.80 . * F 1.30 1.45Glu 616 A . . . . T . −0.17 −0.80 . * F 1.15 0.72 Asp 617 A . . . . T .0.64 −0.11 . * F 0.85 0.61 Gly 618 A . . . . T . −0.17 −0.80 . * F 1.150.63 Leu 619 A . . B . . . 0.06 −0.51 . * . 0.60 0.30 Ile 620 A . . B .. . −0.80 −0.01 . * . 0.30 0.18 Asp 621 . . B B . . . −1.14 0.63 . * .−0.60 0.14 Leu 622 . . B B . . . −1.73 0.63 * * . −0.60 0.16 Ala 623 . .B B . . . −2.20 0.44 . . . −0.60 0.24 Val 624 A . . B . . . −1.73 0.44 *. . −0.60 0.12 Gly 625 A A . . . . . −0.84 0.87 * * . −0.60 0.14 Ala 626A A . . . . . −1.43 0.59 . . . −0.60 0.22 Leu 627 A A . . . . . −1.480.59 . . . −0.60 0.30 Gly 628 A A . . . . . −1.78 0.59 . . . −0.60 0.23Asn 629 . A B B . . . −1.73 0.84 . . . −0.60 0.16 Ala 630 . A B B . . .−1.68 1.03 . . . −0.60 0.16 Val 631 . A B B . . . −1.39 1.26 * . . −0.600.17 Ile 632 . A B B . . . −0.47 1.21 * . . −0.60 0.14 Leu 633 . A B B .. . −0.33 0.81 * . . −0.60 0.27 Trp 634 . A B B . . . −1.19 0.74 * . .−0.60 0.57 Ser 635 . A B B . . . −1.46 0.74 * . . −0.60 0.60 Arg 636 . .B B . . . −0.60 0.70 * * F −0.45 0.54 Pro 637 . . B B . . . −0.600.41 * * . −0.60 0.89 Val 638 . . B B . . . 0.21 0.19 * * . −0.30 0.46Val 639 . . B B . . . −0.09 0.20 * * . −0.30 0.38 Gln 640 . . B B . . .−0.09 0.70 * * . −0.60 0.25 Ile 641 . . B B . . . −1.01 0.66 * * . −0.600.45 Asn 642 . . B . . T . −0.83 0.70 . * . −0.20 0.50 Ala 643 . . B . .T . −0.68 0.56 . * . −0.20 0.39 Ser 644 . . B . . T . 0.18 0.94 . * .−0.20 0.48 Leu 645 A . . . . T . −0.03 0.26 . * . 0.10 0.52 His 646 A .. . . . . 0.56 0.29 . * . 0.18 0.80 Phe 647 A . . . . . . 0.60 0.17 . *. 0.46 0.80 Glu 648 A . . . . T . 0.30 −0.21 . * F 1.84 1.94 Pro 649 A .. . . T . 0.60 −0.21 . * F 1.97 1.00 Ser 650 . . . . T T . 0.52 −0.31. * F 2.80 1.85 Lys 651 A . . . . T . −0.14 −0.41 . . F 1.97 0.75 Ile652 A . . B . . . 0.52 0.37 * * F 0.69 0.42 Asn 653 A . . B . . . 0.630.44 * * . −0.04 0.43 Ile 654 A . . B . . . 0.84 0.06 * * . −0.02 0.42Phe 655 . . B B . . . 0.48 0.06 * * . 0.04 1.00 His 656 . . B . . T .0.48 −0.06 * * . 1.38 0.33 Arg 657 . . B . . T . 1.48 −0.46 * * . 1.720.95 Asp 658 . . . . T T . 1.18 −1.14 * . F 3.06 2.14 Cys 659 . . . . TT . 1.72 −1.54 * . F 3.40 2.11 Lys 660 . . . . T . . 2.53 −1.61 * . F2.86 1.07 Arg 661 . . . . T T . 2.57 −1.61 * . F 3.03 1.25 Ser 662 . . .. T T . 1.87 −1.61 * . F 3.00 3.90 Gly 663 . . . . T T . 1.56 −1.69 * .F 2.97 1.97 Arg 664 . . . . T T . 1.56 −1.20 * . F 2.94 1.45 Asp 665 . .. . T T . 0.70 −0.63 * . F 3.10 0.58 Ala 666 . . B . . T . 0.00 −0.33 *. F 2.09 0.48 Thr 667 . . B . . T . −0.29 −0.26 . . . 1.63 0.25 Cys 668. . B . . T . −0.64 0.24 * . . 0.72 0.15 Leu 669 A A . . . . . −1.571.03 * . . −0.29 0.13 Ala 670 A A . . . . . −2.23 1.21 . . . −0.60 0.07Ala 671 A A . . . . . −2.34 1.30 . . . −0.60 0.07 Phe 672 A A . . . . .−2.34 1.51 . . . −0.60 0.08 Leu 673 A A . . . . . −1.89 1.31 . . . −0.600.11 Cys 674 . A B . . . . −1.97 1.24 . . . −0.60 0.17 Phe 675 . . B B .. . −2.08 1.43 . . . −0.60 0.14 Thr 676 . . B B . . . −2.30 1.43 . . .−0.60 0.14 Pro 677 . . B B . . . −2.19 1.43 . . . −0.60 0.22 Ile 678 . A. B T . . −1.59 1.36 . . . −0.20 0.26 Phe 679 . A B B . . . −0.96 1.00 .. . −0.60 0.28 Leu 680 . A B B . . . −0.96 1.01 . . . −0.60 0.24 Ala 681. A . B . . C −0.64 1.37 . . . −0.40 0.30 Pro 682 . A . B . . C −0.741.09 . . . −0.40 0.60 His 683 . A . B T . . −0.17 0.79 . . . −0.05 1.05Phe 684 . A . B T . . 0.22 0.59 . . . −0.05 1.51 Gln 685 . A B B . . .0.18 0.57 . . F −0.30 1.41 Thr 686 . A B B . . . 0.42 0.79 . . F −0.450.77 Thr 687 . . B B . . . −0.26 0.71 * * F −0.45 0.88 Thr 688 . . B B .. . −0.11 0.61 * * F −0.45 0.35 Val 689 . . B B . . . 0.34 0.21 . * .−0.30 0.48 Gly 690 . . B B . . . 0.34 0.49 . * . −0.60 0.52 Ile 691 . .B B . . . 0.07 0.40 . * . −0.60 0.58 Arg 692 . . B B . . . 0.07 0.41 . *. −0.60 0.79 Tyr 693 . . B B . . . −0.22 0.26 . * . −0.15 1.16 Asn 694 .A B B . . . 0.63 0.44 . * . −0.45 1.63 Ala 695 . A B B . . . 0.98−0.24 * * . 0.45 1.39 Thr 696 . A B B . . . 1.98 −0.24 . * . 0.45 1.54Met 697 . A B B . . . 1.98 −1.00 . * F 0.90 1.87 Asp 698 . A B . . . .1.98 −1.40 . . F 0.90 3.63 Glu 699 . A B . . . . 1.67 −1.14 . . F 1.203.94 Arg 700 . A B . . . . 2.04 −1.14 * * F 1.50 5.75 Arg 701 . A . . T. . 2.47 −1.33 * * F 2.20 5.33 Tyr 702 . A . . T . . 2.48 −1.33 * * F2.50 6.02 Thr 703 . . . . . T C 2.44 −0.83 . * F 3.00 3.11 Pro 704 . . .. . T C 1.63 −0.33 . * F 2.40 2.16 Arg 705 . . B . . T . 1.52 0.36 . * .1.15 1.14 Ala 706 . . B . . T . 1.41 −0.40 * * . 1.45 1.31 His 707 . . B. . . . 1.31 −0.89 . * . 1.59 1.47 Leu 708 . . B . . . . 1.28 −0.89 * *F 1.63 0.74 Asp 709 . . B . . T . 1.49 −0.46 . * F 1.87 0.73 Glu 710 . .. . T T . 1.49 −0.96 * * F 2.91 0.89 Gly 711 . . . . T T . 1.38−1.46 * * F 3.40 2.12 Gly 712 . . . . T T . 1.10 −1.36 * . F 3.06 1.10Asp 713 . . . . T . . 1.91 −0.87 * * F 2.37 0.92 Arg 714 A . . . . . .2.02 −0.47 * * F 1.48 1.49 Phe 715 A . . . . . . 1.43 −0.90 * * F 1.442.95 Thr 716 . . B . . . . 0.92 −0.83 * * F 1.10 1.79 Asn 717 . A B . .. . 0.46 −0.19 * * F 0.45 0.68 Arg 718 . A B . . . . −0.36 0.50 * * F−0.45 0.64 Ala 719 . A B . . . . −0.77 0.40 * * . −0.60 0.37 Val 720 . AB . . . . −0.37 0.30 . . . −0.06 0.31 Leu 721 . A B . . . . −0.40 0.29 .. . 0.18 0.21 Leu 722 . A B . . . . −0.40 0.71 . . F 0.27 0.21 Ser 723 .. . . . T C −0.51 0.61 . . F 1.11 0.48 Ser 724 . . . . . T C −0.73 −0.03. . F 2.40 1.01 Gly 725 . . . . . T C −0.54 −0.03 * . F 2.16 1.01 Gln726 A . . . . T . 0.27 −0.14 * . F 1.57 0.40 Glu 727 A A . . . . . 1.19−0.53 * * F 1.23 0.52 Leu 728 A A . . . . . 0.60 −0.91 * * F 1.14 1.03Cys 729 A A . . . . . 0.90 −0.66 * * . 0.60 0.42 Glu 730 A A . . . . .0.54 −0.66 * * . 0.60 0.39 Arg 731 A A . . . . . 0.51 0.13 * * . −0.300.41 Ile 732 A A . . . . . −0.34 −0.06 * * . 0.45 1.03 Asn 733 A A . . .. . −0.34 0.01 * * . −0.30 0.44 Phe 734 A A . . . . . 0.32 0.70 * * .−0.60 0.19 His 735 . A B . . . . 0.01 0.70 * * . −0.60 0.44 Val 736 . AB . . . . −0.69 0.50 * * . −0.60 0.40 Leu 737 . A B . . . . 0.20 0.60. * . −0.60 0.46 Asp 738 A A . . . . . −0.04 −0.19 . . F 0.62 0.57 Thr739 A . . . . T . −0.20 0.07 . . F 0.74 1.20 Ala 740 A . . . . T . −0.120.07 * . F 0.91 1.08 Asp 741 A . . . . T . 0.52 −0.61 * . . 1.83 1.30Tyr 742 . . B . . T . 0.48 −0.19 * . . 1.70 1.39 Val 743 . . B B . . .0.17 −0.03 . . . 1.13 1.02 Lys 744 . . B B . . . −0.22 −0.04 . . F 0.960.88 Pro 745 . . B B . . . 0.07 0.74 * . F −0.11 0.49 Val 746 . . B B .. . −0.79 0.37 * . . −0.13 0.88 Thr 747 . . B B . . . −0.54 0.37 * * .−0.30 0.33 Phe 748 . . B B . . . 0.07 0.37 . * . −0.30 0.37 Ser 749 . .B B . . . −0.28 0.70 . * . −0.60 0.77 Val 750 . . B B . . . −0.88 0.44. * . −0.60 0.72 Glu 751 . . B B . . . −0.02 0.64 . * . −0.60 0.68 Tyr752 . . B . . . . 0.29 −0.14 . * . 0.50 0.88 Ser 753 . . . . . . C 0.78−0.53 . * . 1.49 1.99 Leu 754 . . . . T . . 1.08 −0.74 * . . 2.03 1.78Glu 755 A . . . . . . 1.90 −0.74 * . F 2.12 1.89 Asp 756 A . . . . T .1.56 −1.00 . . F 2.66 1.92 Pro 757 . . . . T T . 1.59 −0.96 . . F 3.402.31 Asp 758 . . . . T T . 1.29 −1.21 . . F 3.06 2.06 His 759 . . . . .T C 1.29 −0.60 . . F 2.52 1.22 Gly 760 . . . . . . C 1.29 0.09 . . F0.93 0.65 Pro 761 . . B . . . . 1.29 −0.34 . . F 0.99 0.65 Met 762 . . B. . . . 1.16 −0.34 * . . 0.50 0.80 Leu 763 . . B . . . . 0.87 −0.41 * .F 0.89 0.80 Asp 764 . . . . T T . 0.69 0.07 . . F 1.13 0.54 Asp 765 . .. . T T . 0.72 0.07 . . F 1.37 0.85 Gly 766 . . . . T T . 0.62 −0.06 . .F 2.36 1.48 Trp 767 . . . . . T C 0.41 −0.26 * * F 2.40 1.28 Pro 768 . .. B . . C 1.33 0.43 * * F 0.71 0.63 Thr 769 . . B B . . . 0.48 0.43 * *F 0.42 1.25 Thr 770 . . B B . . . 0.18 0.64 * * F 0.03 0.89 Leu 771 . .B B . . . −0.33 0.11 . * . −0.06 0.77 Arg 772 . . B B . . . −0.260.33 * * . −0.30 0.39 Val 773 . . B B . . . −0.74 0.27 * * . −0.30 0.42Ser 774 . . B B . . . −0.72 0.57 * * . −0.60 0.44 Val 775 . . B B . . .−0.41 0.80 * * . −0.60 0.24 Pro 776 . . B B . . . 0.06 1.20 * * . −0.600.52 Phe 777 . . . B T . . −0.72 0.99 * * . −0.20 0.38 Trp 778 . . . . TT . 0.13 1.17 . . . 0.20 0.28 Asn 779 . . . . . T C 0.43 0.93 . . . 0.000.29 Gly 780 . . . . T T . 1.29 0.50 . . . 0.20 0.57 Cys 781 . . . . T T. 1.50 −0.29 . . F 1.25 0.91 Asn 782 . A . . T . . 2.17 −1.20 . . F 1.150.98 Glu 783 . A . . T . . 1.79 −1.10 . . F 1.30 1.35 Asp 784 . A . . T. . 0.93 −0.96 . . F 1.30 1.35 Glu 785 . A . . T . . 1.07 −0.89 . . F1.15 0.62 His 786 . A . . T . . 1.73 −0.86 * . . 1.00 0.56 Cys 787 A A .. . . . 0.92 −0.86 * . . 0.60 0.56 Val 788 A . . . . . . 0.07 −0.17 . .. 0.50 0.26 Pro 789 A . . . . . . −0.74 0.47 . . F −0.25 0.14 Asp 790 AA . . . . . −0.74 0.66 * . F −0.45 0.22 Leu 791 A A . . . . . −1.300.09 * * . −0.30 0.50 Val 792 A A . . . . . −0.52 −0.06 * * . 0.30 0.33Leu 793 A A . . . . . 0.03 −0.49 * * . 0.30 0.38 Asp 794 . A B . . . .0.24 −0.10 . * . 0.30 0.62 Ala 795 A A . . . . . −0.57 −0.79 . * F 0.901.40 Arg 796 A . . . . T . 0.03 −0.74 . * F 1.30 1.40 Ser 797 A . . . .T . 0.58 −1.00 . * F 1.30 1.29 Asp 798 A . . . . T C 0.80 −0.51 . * F1.50 1.85 Leu 799 . . . . . T C 0.20 −0.51 * * F 1.35 0.95 Pro 800 . A .. . . C 0.79 0.10 * * F 0.05 0.70 Thr 801 A A . . . . . 0.43 −0.29 . * .0.30 0.73 Ala 802 A A . . . . . 0.07 0.47 . . . −0.45 1.39 Met 803 A A .. . . . 0.07 0.36 * . . −0.30 0.48 Glu 804 A A . . . . . 0.99 0.33 * * .−0.30 0.58 Tyr 805 A . . B . . . 0.34 −0.16 * . . 0.45 1.12 Cys 806 A .. B . . . −0.16 −0.01 * . . 0.30 0.84 Gln 807 A . . B . . . 0.54 0.06 *. . −0.30 0.40 Arg 808 A . . B . . . 1.19 0.06 * . . −0.30 0.50 Val 809A . . B . . . 0.98 −0.70 * . . 0.75 1.86 Leu 810 . . B B . . . 0.63−0.84 * . F 1.20 1.66 Arg 811 . . B B . . . 1.30 −0.74 * . F 1.35 0.86Lys 812 . . B B . . . 1.30 −0.34 * . F 1.50 2.00 Pro 813 . . . . T . .0.52 −0.99 * . F 2.70 4.06 Ala 814 . . . . T . . 1.08 −1.10 * . F 3.001.11 Gln 815 . . B . . T . 1.30 −0.71 * . F 2.35 0.74 Asp 816 . . B . .T . 0.94 −0.21 * . F 1.75 0.49 Cys 817 . . B . . T . 0.59 0.11 . . .0.70 0.75 Ser 818 . . B . . T . −0.01 0.10 . . . 0.40 0.63 Ala 819 . . BB . . . 0.28 0.39 . . . −0.30 0.31 Tyr 820 . . B B . . . −0.42 0.77 . .. −0.60 0.78 Thr 821 . . B B . . . −0.42 0.99 . . . −0.60 0.50 Leu 822 .. B B . . . −0.07 0.60 . . . −0.60 0.83 Ser 823 . . B B . . . −0.08 0.59. * . −0.60 0.76 Phe 824 . . B B . . . −0.34 0.31 . * . −0.30 0.76 Asp825 . . B B . . . −0.80 0.47 . * F −0.45 0.69 Thr 826 . . B B . . .−1.38 0.57 . * F −0.45 0.44 Thr 827 . . B B . . . −1.46 0.87 . * F −0.450.36 Val 828 . . B B . . . −1.16 0.77 . . . −0.60 0.15 Phe 829 . . B B .. . −0.76 0.77 . . . −0.60 0.18 Ile 830 . . B B . . . −1.07 0.67 . . .−0.60 0.17 Ile 831 . . B B . . . −0.64 0.67 . . . −0.60 0.33 Glu 832 A .. B . . . −0.33 0.03 . * F −0.15 0.74 Ser 833 A . . . . T . 0.63 −0.36. * F 1.00 1.83 Thr 834 A . . . . T . 0.48 −1.04 . * F 1.30 5.10 Arg 835A . . . . T . 0.78 −1.09 . * F 1.30 2.19 Gln 836 A . . . . T . 0.81−0.59 . * F 1.30 1.65 Arg 837 A A . . . . . 0.81 −0.33 . * F 0.45 0.85Val 838 . A B . . . . 0.52 −0.81 . * . 0.60 0.75 Ala 839 . A B . . . .0.52 −0.31 . * . 0.30 0.44 Val 840 . A B . . . . −0.40 −0.23 . * . 0.300.32 Glu 841 A A . . . . . −0.40 0.46 . * . −0.60 0.36 Ala 842 A A . . .. . −0.51 −0.19 . * . 0.30 0.61 Thr 843 A A . . . . . 0.46 −0.29 . * .0.45 1.33 Leu 844 A A . . . . . 0.70 −0.93 . * F 0.90 1.50 Glu 845 A A .. . . . 1.56 −0.50 . * F 0.60 1.47 Asn 846 A . . . . T . 1.56 −1.00 . *F 1.60 1.77 Arg 847 A . . . . T . 1.56 −1.09 * * F 1.90 3.45 Gly 848 A .. . . T . 1.62 −1.27 * * F 2.20 2.01 Glu 849 A . . . . T . 2.13−0.51 * * F 2.50 1.96 Asn 850 . . . . . T C 1.82 −0.53 * . F 3.00 1.34Ala 851 A . . . . T . 0.97 −0.04 * . F 2.20 1.95 Tyr 852 . . B . . T .0.04 0.17 * . . 1.00 0.84 Ser 853 . . B . . T . 0.39 0.86 * . . 0.400.43 Thr 854 . . B B . . . −0.50 0.86 * . . −0.30 0.68 Val 855 . . B B .. . −0.80 1.04 * . . −0.60 0.31 Leu 856 . . B B . . . −0.21 0.67 * . .−0.60 0.31 Asn 857 . . B B . . . −0.27 0.69 * . . −0.60 0.37 Ile 858 . .B B . . . −0.56 0.59 * . F −0.45 0.66 Ser 859 . . B . . . . −0.24 0.44. * F −0.25 0.81 Gln 860 . . B . . . C −0.20 0.16 . * F 0.25 0.81 Ser861 . . . . . T C 0.61 0.44 . * F 0.15 0.96 Ala 862 . . B . . T . −0.090.16 . * F 0.40 1.23 Asn 863 . . B . . T . 0.21 0.56 . * . −0.20 0.62Leu 864 A . . . . T . 0.21 0.66 . . . −0.20 0.47 Gln 865 A A . . . . .−0.60 0.66 . * . −0.60 0.62 Phe 866 A A . . . . . −1.19 0.84 . * . −0.600.32 Ala 867 A A . . . . . −0.60 1.13 * * . −0.60 0.27 Ser 868 A A . . .. . −0.56 0.84 * * . −0.60 0.27 Leu 869 A A . . . . . 0.26 0.44 * . .−0.60 0.62 Ile 870 A A . . . . . 0.26 −0.34 * . . 0.45 1.07 Gln 871 A A. . . . . 0.66 −0.84 * . F 1.24 1.33 Lys 872 A A . . . . . 1.24 −0.84 *. F 1.58 2.16 Glu 873 A A . . . . . 1.20 −1.53 * . F 1.92 5.14 Asp 874 .. . . T T . 1.71 −1.79 . * F 3.06 2.94 Ser 875 . . . . T T . 1.71 −1.80. * F 3.40 1.97 Asp 876 . . . . T T . 1.71 −1.11 . * F 2.91 0.80 Gly 877. . . . T T . 1.00 −1.11 . * F 2.57 0.83 Ser 878 A A . . . . . 0.14−0.54 . . F 1.43 0.33 Ile 879 A A . . . . . 0.14 −0.29 . * . 0.64 0.15Glu 880 A A . . . . . 0.44 0.11 . . . −0.30 0.24 Cys 881 . A B . . . .0.44 −0.31 . * . 0.30 0.31 Val 882 A A . . . . . 0.90 −0.70 * . . 0.600.76 Asn 883 A A . . . . . 1.31 −1.39 * . F 0.75 0.86 Glu 884 A A . . .. . 1.39 −1.39 * * F 0.90 3.15 Glu 885 A A . . . . . 1.39 −1.27 * . F0.90 3.50 Arg 886 A A . . . . . 2.10 −1.51 * . F 0.90 3.77 Arg 887 A A .. . . . 2.96 −1.91 * * F 0.90 4.35 Leu 888 A A . . . . . 2.10 −1.51 * .F 0.90 4.35 Gln 889 A A . . . . . 1.43 −0.87 * . F 0.90 1.65 Lys 890 A A. . . . . 1.43 −0.30 * . F 0.45 0.45 Gln 891 . A B . . . . 0.47 0.10 * .. −0.30 0.88 Val 892 . A B . . . . 0.06 0.06 * * . −0.30 0.38 Cys 893 .A B . . . . 0.62 0.04 * . . −0.30 0.25 Asn 894 . . B . . T . 0.41 0.80 *. . −0.20 0.23 Val 895 . . B . . T . −0.33 0.83 * . . −0.20 0.48 Ser 896. . B . . T . −1.03 0.97 * . . −0.20 0.77 Tyr 897 . . B . . T . −0.071.19 * . . −0.20 0.41 Pro 898 . . B B . . . 0.01 0.79 * * . −0.45 1.09Phe 899 A A . B . . . 0.06 0.64 * * . −0.60 0.82 Phe 900 A A . B . . .0.32 0.26 * * . −0.15 1.05 Arg 901 A A . B . . . 0.67 0.00 * * . −0.300.69 Ala 902 A A . . . . . 0.06 −0.43 * * . 0.45 1.59 Lys 903 A A . . .. . −0.32 −0.57 * * F 0.90 1.36 Ala 904 A A . . . . . −0.32 −0.86 * * F0.75 0.70 Lys 905 A A . B . . . 0.49 −0.07 * * . 0.30 0.60 Val 906 A A .B . . . −0.43 −0.57 * * . 0.60 0.59 Ala 907 A A . B . . . 0.16 0.11 . *. −0.30 0.48 Phe 908 A A . B . . . −0.59 −0.39 . * . 0.30 0.40 Arg 909 AA . B . . . 0.00 0.40 * * . −0.60 0.47 Leu 910 A A . . . . . −0.74−0.24 * * . 0.30 0.80 Asp 911 A A . . . . . −0.19 0.04 * * . −0.30 0.80Phe 912 A A . . . . . 0.44 −0.36 * * . 0.30 0.55 Glu 913 A A . . . . .0.84 −0.36 * * . 0.45 1.33 Phe 914 A A . . . . . −0.16 −0.66 * * . 0.751.07 Ser 915 A . . . . T . −0.04 0.03 . * F 0.25 0.87 Lys 916 A . . . .T . −0.86 0.03 . * F 0.25 0.43 Ser 917 A . . . . T . −0.19 0.71 . . .−0.20 0.41 Ile 918 A . . . . T . −0.22 0.43 . . . −0.20 0.42 Phe 919 A A. . . . . −0.33 0.54 . . . −0.60 0.28 Leu 920 A A . . . . . −0.03 1.23 .. . −0.60 0.18 His 921 A A . . . . . −0.97 0.84 . * . −0.60 0.43 His 922A A . . . . . −0.67 0.84 . * . −0.60 0.35 Leu 923 A A . . . . . −0.590.06 . * . −0.30 0.74 Glu 924 A A . . . . . −0.48 0.06 . * . −0.30 0.45Ile 925 A A . . . . . −0.26 0.06 . * . −0.30 0.33 Glu 926 A A . . . . .−0.57 0.06 . * . −0.30 0.41 Leu 927 A A . . . . . −0.83 −0.20 . * . 0.300.23 Ala 928 A A . . . . . −0.02 0.19 . * . −0.30 0.44 Ala 929 A A . . .. . −0.32 −0.50 . * . 0.30 0.43 Gly 930 A . . . . T . 0.57 −0.11 . * F0.85 0.69 Ser 931 . . . . . T C 0.57 −0.40 * * F 1.20 1.11 Asp 932 . . .. . T C 1.49 −0.90 . . F 1.50 1.89 Ser 933 . . . . . T C 2.08 −1.40 . .F 1.84 3.75 Asn 934 . . . . . . C 2.37 −1.83 . . F 1.98 4.67 Glu 935 A .. . . . . 2.40 −1.83 * * F 2.12 3.75 Arg 936 A . . . . . . 2.74 −1.34 .. F 2.46 4.04 Asp 937 . . . . T T . 2.74 −1.73 . * F 3.40 5.02 Ser 938 .. . . . T C 3.04 −2.13 . . F 2.86 5.02 Thr 939 A . . . . T . 3.04 −2.13. . F 2.32 4.28 Lys 940 A . . . . T . 2.19 −1.73 * . F 1.98 4.12 Glu 941A A . . . . . 1.49 −1.09 * . F 1.24 2.28 Asp 942 A A . . . . . 1.28−0.97 . . F 0.90 1.60 Asn 943 A A . . . . . 0.77 −1.03 . * F 0.90 1.24Val 944 A A . . . . . 1.19 −0.34 . * . 0.30 0.59 Ala 945 A A . . . . .0.44 −0.34 . * . 0.30 0.69 Pro 946 A A . . . . . 0.41 0.44 . * . −0.600.37 Leu 947 A A . . . . . −0.40 0.54 . * . −0.60 0.68 Arg 948 A A . . .. . −0.36 0.59 . * . −0.60 0.56 Phe 949 A A . . . . . 0.26 0.09 . * .−0.30 0.72 His 950 A A . . . . . 0.84 0.41 . * . −0.45 1.37 Leu 951 A A. . . . . 0.47 −0.27 * * . 0.45 1.21 Lys 952 A A . . . . . 1.28 0.23 * *. −0.15 1.41 Tyr 953 A A . . . . . 0.31 −0.56 * * . 0.75 1.73 Glu 954 AA . . . . . 0.20 −0.41 . * . 0.45 1.56 Ala 955 A . . B . . . −0.47 −0.41. * . 0.30 0.64 Asp 956 A . . B . . . 0.03 0.37 . * . −0.30 0.35 Val 957A . . B . . . 0.10 0.10 . * . −0.30 0.30 Leu 958 A . . B . . . 0.04 0.10. . . −0.30 0.57 Phe 959 A . . B . . . −0.26 −0.01 . . . 0.30 0.46 Thr960 A . . B . . . 0.03 0.37 . . F 0.06 0.83 Arg 961 A . . B . . . −0.780.11 . . F 0.42 1.35 Ser 962 . . . . T T . −0.22 0.11 . . F 1.43 1.29Ser 963 . . . . . T C 0.56 −0.29 . . F 2.04 1.19 Ser 964 . . . . . T C1.01 −0.27 . . F 2.10 0.83 Leu 965 . . . . . T C 1.32 0.49 . . F 0.990.97 Ser 966 . . . . . . C 0.36 0.10 . * . 0.88 1.25 His 967 . . B . . .. 0.70 0.36 . * . 0.32 0.69 Tyr 968 . . B . . . . 0.19 −0.03 . * . 0.861.68 Glu 969 . . B . . . . 0.49 −0.03 . * . 0.65 1.04 Val 970 A . . . .. . 1.00 −0.01 . * . 0.65 1.22 Lys 971 A . . . . . . 1.00 −0.13 . * F0.80 1.05 Leu 972 A . . . . . . 0.22 −0.50 . * F 0.65 0.81 Asn 973 . . .. . T C 0.47 0.19 * * F 0.45 0.90 Ser 974 A . . . . T . 0.58 −0.46 * * F0.85 0.78 Ser 975 A . . . . T . 1.19 −0.46 * * F 1.00 1.85 Leu 976 . . B. . T . 1.14 −0.39 * * F 1.31 1.80 Glu 977 . . B . . . . 1.61 −0.79 * .F 1.72 2.25 Arg 978 . . B . . T . 0.72 −0.74 * . F 2.23 1.66 Tyr 979 . .B . . T . 0.68 −0.44 . . F 2.24 1.41 Asp 980 . . . . T T . 0.77 −0.70 .. F 3.10 0.81 Gly 981 . . . . T T . 1.37 −0.27 * . F 2.49 0.64 Ile 982 .. . . T . . 0.67 0.16 * . F 1.38 0.63 Gly 983 . . . . . . C 0.26 0.19. * F 0.87 0.33 Pro 984 . . . . . T C −0.17 0.57 * . F 0.46 0.44 Pro 985. . . . T T . −1.06 0.71 * . F 0.35 0.34 Phe 986 . . . . T T . −1.410.71 * * . 0.20 0.24 Ser 987 . . B . . T . −0.41 1.07 * * . −0.20 0.13Cys 988 . . B B . . . −0.96 0.64 * * . −0.60 0.17 Ile 989 . . B B . . .−0.74 0.90 * * . −0.60 0.14 Phe 990 . . B B . . . −0.53 0.51 * * . −0.600.18 Arg 991 . . B B . . . −0.64 0.53 * * . −0.60 0.53 Ile 992 . . B B .. . −0.69 0.64 * * . −0.60 0.63 Gln 993 . . B B . . . −0.83 0.39 * * .−0.30 0.72 Asn 994 . . . B T . . −0.64 0.29 * * . 0.10 0.30 Leu 995 . .. B T . . −0.16 1.07 . * . −0.20 0.37 Gly 996 . . . B T . . −1.160.81 * * . −0.20 0.33 Leu 997 . . . . . . C −0.30 1.10 . . . −0.20 0.14Phe 998 . . B . . . . −0.64 1.20 . . . −0.40 0.24 Pro 999 . . B . . . .−1.53 0.94 . . . −0.40 0.24 Ile 1000 A . . B . . . −1.32 1.20 . . .−0.60 0.20 His 1001 A . . B . . . −1.58 1.13 . . . −0.60 0.23 Gly 1002 A. . B . . . −0.72 0.96 * . . −0.60 0.15 Ile 1003 A . . B . . . −0.910.53 . * . −0.60 0.42 Met 1004 A . . B . . . −1.01 0.53 . * . −0.60 0.22Met 1005 . . B B . . . −1.01 0.51 . * . −0.60 0.32 Lys 1006 . . B B . .. −1.19 0.77 . * . −0.60 0.32 Ile 1007 . . B B . . . −1.73 0.51 . * .−0.60 0.50 Thr 1008 . . B B . . . −1.43 0.59 . * . −0.60 0.35 Ile 1009 .. B B . . . −1.14 0.47 * * . −0.60 0.18 Pro 1010 . . B B . . . −0.430.96 * * . −0.60 0.37 Ile 1011 . . B B . . . −0.78 0.27 * * . 0.04 0.50Ala 1012 . . B B . . . −0.23 0.17 * * . 0.38 0.95 Thr 1013 . . B . . T .0.08 −0.09 * * F 1.87 0.61 Arg 1014 . . . . T T . 1.08 −0.11 * * F 2.761.40 Ser 1015 . . . . T T . 0.48 −0.80 * * F 3.40 2.71 Gly 1016 . . . .T T . 0.56 −0.61 * * F 3.06 1.55 Asn 1017 . A . . T . . 1.19 −0.41 * . F1.87 0.65 Arg 1018 . A B . . . . 0.69 −0.41 * * F 1.13 0.97 Leu 1019 . AB . . . . 0.69 −0.11 * * F 0.79 0.81 Leu 1020 . A B . . . . 0.99 −0.54 .. F 0.75 0.99 Lys 1021 . A B . . . . 0.63 −0.94 * . F 0.75 0.84 Leu 1022. A B . . . . −0.18 −0.16 * . F 0.45 0.88 Arg 1023 . A B . . . . −0.60−0.16 * . F 0.45 0.88 Asp 1024 . A B . . . . 0.21 −0.36 * * F 0.45 0.64Phe 1025 . A B . . . . 1.02 −0.36 * * . 0.45 1.29 Leu 1026 A A . . . . .0.12 −1.04 * * F 0.90 1.14 Thr 1027 A A . . . . . 0.34 −0.40 * . F 0.450.51 Asp 1028 A A . . . . . 0.23 0.10 . . F −0.15 0.59 Glu 1029 A A . .. . . −0.08 −0.29 * . . 0.45 1.15 Val 1030 A A . . . . . 0.32 −0.49 . .. 0.45 1.15 Ala 1031 A A . . . . . 0.47 −0.59 * . . 0.60 0.93 Asn 1032 .. . . T T . 0.78 −0.01 * . F 1.25 0.29 Thr 1033 . . . . T T . −0.110.39 * . F 0.65 0.62 Ser 1034 . . B . . T . −0.40 0.43 * . F −0.05 0.43Cys 1035 . . B . . T . 0.11 0.84 * * . −0.20 0.28 Asn 1036 . . . . T . .0.70 0.87 . * . 0.16 0.19 Ile 1037 . . . . T . . 0.40 0.79 . * . 0.320.23 Trp 1038 . . . . T T . 0.40 0.79 . . . 0.68 0.58 Gly 1039 . . . . .T C 0.70 0.70 . . F 0.79 0.52 Asn 1040 . . . . T T . 1.12 0.30 . * F1.60 1.28 Ser 1041 . . . . . T C 1.23 0.37 . * F 1.24 1.91 Thr 1042 . .. . . . C 1.91 −0.54 . * F 1.78 3.79 Glu 1043 . . . . T . . 1.89 −0.54. * F 1.82 3.64 Tyr 1044 . . . . T . . 2.02 −0.46 . . F 1.66 3.92 Arg1045 . . . . . T C 1.17 −0.41 . . F 1.80 4.20 Pro 1046 . . . . . T C1.47 −0.26 . . F 2.10 1.80 Thr 1047 . . . . . T C 1.78 −0.26 . . F 2.401.99 Pro 1048 . . . . . T C 1.78 −1.01 . * F 3.00 1.76 Val 1049 A A . .. . . 1.21 −1.01 . * F 2.10 1.90 Glu 1050 A A . . . . . 1.21 −0.76 * . F1.80 1.09 Glu 1051 A A . . . . . 1.53 −1.24 * . F 1.50 1.38 Asp 1052 A A. . . . . 1.26 −1.67 * . F 1.20 3.63 Leu 1053 A A . . . . . 1.26 −1.81 *. F 0.90 2.12 Arg 1054 A A . . . . . 2.11 −1.39 * * F 0.90 1.89 Arg 1055A A . . . . . 1.30 −0.99 * * F 0.90 1.96 Ala 1056 A A . . . . . 1.30−0.30 * . F 0.60 1.96 Pro 1057 A A . . . . . 1.27 −0.59 * * F 0.90 1.61Gln 1058 A A . . . . . 1.78 −0.09 * . F 0.60 1.12 Leu 1059 . A B . . . .1.67 0.30 * . . 0.13 1.48 Asn 1060 . A . . . . C 1.26 0.20 . . . 0.611.54 His 1061 . . . . . T C 1.84 0.16 . . F 1.44 1.19 Ser 1062 . . . . .T C 1.20 −0.24 . . F 2.32 2.42 Asn 1063 . . . . T T . 0.34 −0.29 * . F2.80 1.12 Ser 1064 . . . . T T . 0.86 −0.04 * . F 2.37 0.61 Asp 1065 . .B B . . . −0.03 −0.16 . . F 1.29 0.61 Val 1066 . . B B . . . 0.00 0.14 .. F 0.41 0.27 Val 1067 . . B B . . . −0.37 0.14 . . . −0.02 0.32 Ser1068 . . B . . T . −0.37 0.33 . * . 0.10 0.10 Ile 1069 . . B . . T .−0.96 0.73 * * . −0.20 0.22 Asn 1070 . . B . . T . −0.84 0.77 . * .−0.20 0.21 Cys 1071 . . B . . T . −0.80 0.13 * * . 0.10 0.31 Asn 1072 .. B B . . . −0.80 0.43 * * . −0.60 0.36 Ile 1073 . . B B . . . −0.710.39 * * . −0.30 0.17 Arg 1074 . . B B . . . 0.18 0.41 * * . −0.60 0.48Leu 1075 . . B B . . . 0.18 0.24 * * . −0.30 0.48 Val 1076 . . B . . T .0.84 0.24 . * F 0.40 1.18 Pro 1077 . . . . . T C −0.04 −0.44 * * F 1.201.05 Asn 1078 . . . . T T . 0.84 0.24 * * F 0.65 0.89 Gln 1079 A . . . .T . 0.03 −0.04 * * F 1.00 1.93 Glu 1080 . A B . . . . 0.81 0.10 . * F0.00 1.08 Ile 1081 . A B . . . . 0.86 0.17 . * F −0.15 0.91 Asn 1082 . AB . . . . 0.26 0.46 . * . −0.60 0.43 Phe 1083 . A B . . . . −0.09 0.74. * . −0.60 0.21 His 1084 . A B . . . . −0.09 1.17 . * . −0.60 0.29 Leu1085 . A B . . . . −0.90 0.89 . * . −0.60 0.29 Leu 1086 . A . . . . C−0.30 1.17 . * . −0.40 0.28 Gly 1087 . A . . T . . −1.11 1.30 * * .−0.20 0.22 Asn 1088 A A . . . . . −0.30 1.49 * * . −0.60 0.22 Leu 1089 AA . . . . . −0.57 0.80 * * . −0.60 0.51 Trp 1090 A A . . . . . −0.570.50 * * . −0.60 0.69 Leu 1091 A A . . . . . 0.29 0.76 * . . −0.60 0.35Arg 1092 A A . . . . . 0.04 0.36 * * . −0.30 0.86 Ser 1093 A A . . . . .−0.77 0.17 * . . −0.30 0.83 Leu 1094 A A . . . . . 0.09 −0.06 * . F 0.450.83 Lys 1095 A A . . . . . 0.13 −0.74 * . F 0.75 0.84 Ala 1096 A A . .. . . 0.99 0.01 * * . −0.30 0.99 Leu 1097 A A . . . . . 0.58 −0.37 * . .0.45 2.39 Lys 1098 A A . . . . . 0.28 −0.67 . . F 0.90 1.60 Tyr 1099 A A. . . . . 1.13 −0.06 * . F 0.60 1.57 Lys 1100 A A . . . . . 0.20 −0.56 *. F 0.90 3.81 Ser 1101 A A . . . . . 0.19 −0.56 . * F 0.90 1.34 Met 1102A A . . . . . 0.14 0.06 . * . −0.30 0.84 Lys 1103 A A . . . . . 0.10−0.06 . * . 0.30 0.31 Ile 1104 . A B . . . . −0.24 0.34 * * . −0.30 0.38Met 1105 A A . . . . . −0.88 0.46 * * . −0.60 0.38 Val 1106 A A . . . .. −1.39 0.34 * * . −0.30 0.19 Asn 1107 A A . . . . . −0.79 1.03 . * .−0.60 0.23 Ala 1108 A A . . . . . −0.72 0.74 . * . −0.60 0.40 Ala 1109 AA . . . . . 0.17 0.13 . * . −0.15 1.05 Leu 1110 A A . . . . . 0.07−0.11 * . . 0.45 1.13 Gln 1111 A A . . . . . 0.89 0.27 * . . −0.30 0.97Arg 1112 A A . . . . . 0.59 0.27 * . . −0.15 1.31 Gln 1113 . A B . . . .0.97 0.16 * . . −0.15 2.13 Phe 1114 . A . . T . . 0.86 −0.10 * . . 0.851.90 His 1115 . A . . . . C 0.78 0.29 * . . −0.10 0.84 Ser 1116 . A . .. . C 0.08 0.97 . * . −0.40 0.34 Pro 1117 . . . B . . C 0.08 1.36 . * .−0.40 0.34 Phe 1118 . A . B . . C 0.08 0.57 . * . −0.40 0.49 Ile 1119 .A B B . . . 0.78 0.07 . * . −0.30 0.63 Phe 1120 . A B B . . . 0.81 −0.31. . . 0.30 0.71 Arg 1121 A A . B . . . 0.90 −0.74 . . F 1.24 1.37 Glu1122 . A . . T . . 0.81 −1.10 * * F 1.98 3.01 Glu 1123 . A . . . . C1.62 −1.40 * * F 2.12 4.66 Asp 1124 . . . . . T C 2.51 −2.19 * . F 2.864.66 Pro 1125 . . . . T T . 2.32 −1.79 * * F 3.40 4.66 Ser 1126 . . . .T T . 1.36 −1.10 * . F 3.06 1.89 Arg 1127 A . . . . T . 0.66 −0.46 . . F1.87 0.84 Gln 1128 A . . B . . . 0.66 0.33 * . F 0.53 0.47 Ile 1129 A .. B . . . −0.23 −0.10 * . . 0.64 0.61 Val 1130 A . . B . . . −0.320.20 * . . −0.30 0.22 Phe 1131 . . B B . . . 0.02 0.59 * . . −0.60 0.17Glu 1132 . . B B . . . −0.09 0.19 * . . −0.30 0.48 Ile 1133 . . B B . .. −0.09 −0.10 * * F 0.60 1.12 Ser 1134 . A . . . . C 0.80 −0.74 . * F1.10 2.24 Lys 1135 . A . . T . . 1.37 −1.53 . . F 1.30 2.16 Gln 1136 . A. . T . . 2.07 −0.61 . * F 1.30 3.24 Glu 1137 A A . . . . . 1.21 −0.90. * F 0.90 4.18 Asp 1138 . A . B T . . 1.89 −0.64 . * F 1.30 1.55 Trp1139 . A . B T . . 1.30 −0.21 . * . 0.85 1.39 Gln 1140 . A B B . . .0.97 0.07 . * . −0.30 0.56 Val 1141 . A B B . . . 0.08 0.99 . * . −0.600.35 Pro 1142 . A B B . . . −0.81 1.67 . * . −0.60 0.24 Ile 1143 . . B B. . . −1.67 1.44 . * . −0.60 0.10 Trp 1144 . . B B . . . −1.72 1.69 . *. −0.60 0.10 Ile 1145 . . B B . . . −2.02 1.47 . . . −0.60 0.06 Ile 1146. . B B . . . −1.48 1.43 . . . −0.60 0.12 Val 1147 . . B B . . . −2.081.23 . . . −0.60 0.16 Gly 1148 . . B B . . . −1.53 1.00 . . F −0.45 0.19Ser 1149 . . . B . . C −1.59 0.74 . . F −0.25 0.27 Thr 1150 . . . B . .C −1.51 0.49 . . F −0.25 0.35 Leu 1151 . . . B . . C −1.43 0.53 . . F−0.25 0.30 Gly 1152 . . . B T . . −1.39 0.79 . . F −0.05 0.18 Gly 1153 .A B B . . . −1.86 1.09 . . . −0.60 0.10 Leu 1154 . A B B . . . −2.141.29 . . . −0.60 0.10 Leu 1155 . A B B . . . −2.64 1.10 . . . −0.60 0.11Leu 1156 A A . B . . . −2.64 1.36 . . . −0.60 0.09 Leu 1157 A A . B . .. −3.16 1.61 . . . −0.60 0.09 Ala 1158 A A . B . . . −3.62 1.57 . . .−0.60 0.08 Leu 1159 A A . B . . . −3.40 1.57 . . . −0.60 0.08 Leu 1160 AA . B . . . −3.40 1.39 . . . −0.60 0.10 Val 1161 A A . B . . . −2.881.39 . . . −0.60 0.08 Leu 1162 A A . B . . . −2.02 1.80 * . . −0.60 0.10Ala 1163 A A . B . . . −2.24 1.11 . . . −0.60 0.25 Leu 1164 A A . B . .. −1.78 1.11 . * . −0.60 0.27 Trp 1165 A A . B . . . −1.67 0.90 . . .−0.60 0.33 Lys 1166 A A . B . . . −1.51 1.00 . . . −0.60 0.28 Leu 1167 AA . B . . . −0.59 1.29 . . . −0.60 0.29 Gly 1168 A . . B . . . −0.300.60 . . . −0.60 0.55 Phe 1169 . . B B . . . −0.08 0.07 * . . −0.30 0.37Phe 1170 . . B B . . . 0.32 0.57 * . . −0.60 0.45 Arg 1171 . . B B . . .0.39 −0.11 * . . 0.30 0.89 Ser 1172 . . . B . . C 1.31 −0.54 * . F 1.102.02 Ala 1173 . . . . . . C 1.77 −1.33 * . F 1.30 4.57 Arg 1174 . . . .. . C 2.47 −2.11 * . F 1.30 4.57 Arg 1175 . . . . T . . 2.96 −2.11 * . F1.84 5.90 Arg 1176 . . . . T . . 2.50 −2.07 * . F 2.18 9.03 Arg 1177 . .. . T . . 1.99 −2.14 . . F 2.52 4.56 Glu 1178 . . . . . T C 2.58 −1.46 .. F 2.86 1.92 Pro 1179 . . . . T T . 2.26 −1.46 . . F 3.40 1.64 Gly 1180. . . . T T . 1.83 −1.03 . . F 3.06 1.29 Leu 1181 . . . . . T C 1.51−0.54 . * F 2.69 1.08 Asp 1182 . . . . . T C 1.44 −0.11 . * F 2.22 1.08Pro 1183 . . . . . T C 0.59 −0.54 * * F 2.35 2.18 Thr 1184 . . . . . T C−0.01 −0.33 * . F 1.88 1.96 Pro 1185 . . B . . T . 0.33 −0.33 * . F 1.700.97 Lys 1186 . A B . . . . 0.76 −0.33 * * F 1.28 1.08 Val 1187 . A B .. . . 0.37 −0.33 * . F 0.96 0.96 Leu 1188 A A . . . . . 0.19 −0.39 * . .0.64 0.79 Glu 1189 A A . . . . . 0.11 −0.39 * . . 0.47 0.51

TABLE VIII Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A A. . . . . −1.47 0.70 . . . −0.60 0.31 Ala 2 A A . . . . . −1.38 0.96 . .. −0.60 0.20 Leu 3 A A . . . . . −1.80 0.91 . . . −0.60 0.21 Met 4 A A .. . . . −2.27 1.17 . . . −0.60 0.17 Leu 5 A A . . . . . −2.69 1.20 . . .−0.60 0.13 Ser 6 A A . . . . . −2.39 1.39 . * . −0.60 0.13 Leu 7 A A . .. . . −2.61 1.09 . . . −0.60 0.17 Val 8 A A . . . . . −2.61 1.16 * . .−0.60 0.17 Leu 9 A A . . . . . −1.97 1.16 * . . −0.60 0.11 Ser 10 A A .. . . . −1.97 0.77 * . . −0.60 0.26 Leu 11 . A B . . . . −2.01 0.77 * .. −0.60 0.28 Leu 12 . A B . . . . −1.50 0.56 * . . −0.60 0.34 Lys 13 . AB . . . . −0.99 0.26 * . F −0.15 0.34 Leu 14 . A . . . . C −0.18 0.30 .. F 0.05 0.41 Gly 15 . . . . T T . −0.17 0.01 * . F 0.65 0.86 Ser 16 . .. . . T C 0.64 0.24 * * F 0.45 0.45 Gly 17 . . . . . T C 0.60 0.64 * . F0.15 0.95 Gln 18 . . B . . T . −0.14 0.60 * . F −0.05 0.71 Trp 19 . . BB . . . 0.32 0.96 . . . −0.60 0.46 Gln 20 . . B B . . . 0.46 1.00 . * .−0.60 0.46 Val 21 . . B B . . . 0.76 1.00 . * . −0.60 0.41 Phe 22 . . BB . . . 1.14 0.60 . * . −0.30 0.65 Gly 23 . . . . . T C 0.93 −0.31 . . .1.50 0.75 Pro 24 . . . . T T . 0.37 −0.29 . . F 2.30 1.57 Asp 25 . . . .. T C 0.37 −0.29 . . F 2.40 1.34 Lys 26 . . . . . T C 0.63 −0.67 * . F3.00 2.35 Pro 27 . . . B . . C 0.52 −0.60 * . F 2.30 1.54 Val 28 . . B B. . . 0.01 −0.34 * . . 1.20 0.76 Gln 29 . . B B . . . −0.12 0.30 * . .0.30 0.28 Ala 30 . . B B . . . −0.12 0.73 . . . −0.30 0.18 Leu 31 . . BB . . . −0.17 0.30 . . . −0.30 0.42 Val 32 . . B B . . . −0.54 −0.34 . .. 0.30 0.41 Gly 33 A . . B . . . −0.28 −0.24 . . F 0.45 0.41 Glu 34 A A. . . . . −0.98 −0.24 . . F 0.45 0.50 Asp 35 A A . . . . . −0.69 −0.14 .. F 0.45 0.58 Ala 36 A A . . . . . −0.54 −0.40 . . . 0.30 0.79 Ala 37 AA . B . . . −0.39 −0.26 . . . 0.30 0.24 Phe 38 A A . B . . . −0.86 0.53. . . −0.60 0.13 Ser 39 A A . B . . . −1.16 1.21 . . . −0.60 0.10 Cys 40A A . B . . . −1.37 1.10 . . . −0.60 0.14 Phe 41 A A . B . . . −0.731.03 . . . −0.60 0.24 Leu 42 . A . B . . C −0.46 0.24 . . . −0.10 0.36Ser 43 . . . . . T C 0.24 0.34 . * F 0.45 0.98 Pro 44 . . . . . T C−0.04 0.17 . * F 0.60 1.82 Lys 45 . . . . . T C 0.62 −0.11 . * F 1.202.23 Thr 46 A . . . . T . 0.73 −0.80 . * F 1.30 2.88 Asn 47 A A . . . .. 0.94 −0.69 . * F 0.90 1.88 Ala 48 A A . . . . . 1.24 −0.50 . * . 0.300.93 Glu 49 A A . . . . . 0.60 −0.50 . * . 0.45 1.12 Ala 50 A A . . . .. 0.67 −0.34 . * . 0.30 0.52 Met 51 A A . . . . . 0.28 −0.74 * * . 0.601.00 Glu 52 A A . . . . . −0.42 −0.46 * . . 0.30 0.50 Val 53 A A . . . .. 0.28 0.33 * . . −0.30 0.43 Arg 54 A A . . . . . −0.07 −0.17 * . . 0.300.85 Phe 55 A A . . . . . 0.52 −0.36 * * . 0.30 0.48 Phe 56 A . . . . T. 0.42 0.04 * * . 0.25 1.13 Arg 57 A . . . . T . 0.12 0.19 * * . 0.100.50 Gly 58 . . . . T T . 0.68 0.57 * . F 0.35 0.77 Gln 59 . . . . T T .−0.29 0.17 * * F 0.80 1.20 Phe 60 . . . B . . C −0.44 0.03 * * F 0.050.45 Ser 61 . . . B . . C 0.22 0.67 * * F −0.25 0.34 Ser 62 . . B B . .. −0.70 0.74 * * . −0.60 0.27 Val 63 . . B B . . . −0.60 1.03 * . .−0.60 0.25 Val 64 . . B B . . . −0.49 1.00 * . . −0.60 0.30 His 65 . . BB . . . 0.21 0.61 * . . −0.26 0.43 Leu 66 . . B B . . . 0.17 0.23 * . .0.38 0.98 Tyr 67 . . B . . T . 0.51 0.01 * . . 1.27 1.30 Arg 68 . . . .T T . 1.37 −0.63 * . F 3.06 1.92 Asp 69 . . . . T T . 2.22 −1.13 * . F3.40 3.88 Gly 70 . . . . T T . 2.04 −1.41 * . F 3.06 4.29 Lys 71 . . . .T . . 2.16 −1.74 * . F 2.52 3.39 Asp 72 . . . . . . C 1.80 −0.96 . . F1.98 1.76 Gln 73 . . . . . . C 1.69 −0.34 . . F 1.34 1.76 Pro 74 . . B .. . . 1.09 −0.37 . . F 0.80 1.52 Phe 75 . . B . . . . 1.22 0.24 . . .−0.10 0.90 Met 76 . . B . . . . 1.18 0.67 . . . −0.40 0.80 Gln 77 . . B. . . . 0.93 0.67 . . . −0.40 0.90 Met 78 . . B . . . . 0.93 1.00 * . .−0.25 1.63 Pro 79 . . B . . . . 0.80 0.61 * * . 0.09 2.85 Gln 80 . . . .T . . 1.61 0.43 * * F 0.98 1.63 Tyr 81 . . . . T T . 1.90 0.03 . * F1.82 3.23 Gln 82 A . . . . T . 1.94 −0.10 . * F 2.36 3.01 Gly 83 . . . .T T . 1.73 −0.53 . * F 3.40 3.48 Arg 84 . . B . . T . 1.09 −0.24 . * F2.36 1.83 Thr 85 . . B . . . . 1.13 −0.36 . * F 1.90 0.78 Lys 86 . . B .. . . 1.38 −0.76 . * F 2.24 1.59 Leu 87 . . B . . . . 1.08 −1.19 * * F2.13 1.35 Val 88 . . B . . T . 0.53 −0.80 . * F 2.22 1.26 Lys 89 . . B .. T . −0.17 −0.60 . . F 2.30 0.44 Asp 90 . . B . . T . 0.14 −0.10 * . F1.77 0.54 Ser 91 . . B . . T . −0.24 −0.79 * . . 1.84 1.26 Ile 92 A A .. . . . 0.68 −1.00 * * . 1.06 0.62 Ala 93 A A . . . . . 0.64 −1.00 . * F0.98 0.73 Glu 94 A A . . . . . 0.30 −0.31 . * F 0.45 0.38 Gly 95 A A . .. . . −0.51 −0.31 * * F 0.45 0.73 Arg 96 A A . . . . . −0.10 −0.31 * * F0.45 0.60 Ile 97 A A . . . . . −0.02 −0.81 . * F 0.75 0.67 Ser 98 A A .. . . . 0.57 −0.13 * * . 0.30 0.56 Leu 99 A A . . . . . 0.57 −0.56 * * .0.60 0.50 Arg 100 A A . . . . . 0.02 −0.16 * * . 0.45 1.14 Leu 101 A A .. . . . −0.40 −0.16 * * . 0.30 0.60 Glu 102 . A B . . . . −0.37 −0.06. * . 0.45 1.04 Asn 103 . A B . . . . −0.88 −0.10 . * . 0.30 0.40 Ile104 . A B . . . . −0.07 0.59 . * . −0.60 0.40 Thr 105 . A B . . . .−0.77 −0.10 . . . 0.30 0.38 Val 106 . A B . . . . −0.30 0.40 . . . −0.600.24 Leu 107 . A B . . . . −1.11 0.43 . . . −0.60 0.34 Asp 108 . A B . .. . −1.36 0.43 . . . −0.60 0.19 Ala 109 . A B . . . . −0.81 0.70 . . .−0.60 0.41 Gly 110 . . . . T . . −1.17 0.49 * . . 0.00 0.49 Leu 111 . .B . . T . −0.20 0.37 * . . 0.10 0.16 Tyr 112 . . B . . T . −0.280.37 * * . 0.10 0.30 Gly 113 . . B . . T . −0.58 0.56 * * . −0.20 0.22Cys 114 . . B . . T . −0.29 0.51 * * . −0.20 0.35 Arg 115 . . B B . . .0.06 0.21 * * . −0.30 0.30 Ile 116 . . B B . . . 0.57 −0.14 * * F 0.450.52 Ser 117 . . B B . . . 0.57 −0.19 * * F 0.76 1.31 Ser 118 . . B . .T . 0.67 0.00 * * F 1.32 1.05 Gln 119 . . B . . T . 1.33 0.76 . * F 0.582.35 Ser 120 . . . . T T . 1.27 0.47 . * F 1.14 3.03 Tyr 121 . . . . T T. 1.57 0.09 . . F 1.60 4.52 Tyr 122 . A . . T . . 0.98 0.20 . . . 0.892.64 Gln 123 . A B . . . . 0.99 0.49 . . . 0.03 1.38 Lys 124 . A B . . .. 0.99 1.01 * . . −0.28 0.93 Ala 125 . A B . . . . 0.48 0.26 * . . 0.011.02 Ile 126 . A B . . . . 0.72 0.19 . * . −0.30 0.49 Trp 127 . A B . .. . 0.11 0.19 . * . −0.30 0.42 Glu 128 A A . . . . . −0.19 0.83 * * .−0.60 0.31 Leu 129 A A . . . . . −0.82 0.71 * * . −0.60 0.59 Gln 130 . AB . . . . −1.04 0.53 . * . −0.60 0.57 Val 131 . A B . . . . −0.50 0.30. * . −0.30 0.27 Ser 132 . A . . . . C −0.51 0.73 . * . −0.40 0.33 Ala133 . A . . . . C −1.37 0.43 . * . −0.40 0.25 Leu 134 . A B . . . .−0.77 0.67 . * . −0.60 0.25 Gly 135 . A . . T . . −1.58 0.46 . . . −0.200.29 Ser 136 . . B B . . . −1.61 0.76 . . . −0.60 0.24 Val 137 . . B B .. . −1.61 0.94 . . . −0.60 0.20 Pro 138 . . B B . . . −1.91 0.64 . . .−0.60 0.27 Leu 139 . . B B . . . −1.69 0.90 . . . −0.60 0.14 Ile 140 . .B B . . . −1.69 1.01 . . . −0.60 0.19 Ser 141 . . B B . . . −1.63 0.80 .. . −0.60 0.12 Ile 142 . . B B . . . −1.63 1.13 . . . −0.60 0.24 Ala 143. . B B . . . −1.42 1.09 * . . −0.60 0.25 Gly 144 . . B B . . . −0.500.40 * . . −0.34 0.31 Tyr 145 . . B B . . . 0.39 0.01 * * . 0.22 0.87Val 146 . . B B . . . −0.20 −0.67 * . . 1.53 1.44 Asp 147 . . B . . T .0.69 −0.49 * * F 2.04 1.02 Arg 148 . . B . . T . 0.47 −0.51 * . F 2.601.13 Asp 149 . . B . . T . 0.00 −0.59 * . F 2.34 1.25 Ile 150 . . B . .T . −0.42 −0.54 * . . 1.78 0.62 Gln 151 . A B . . . . 0.43 0.03 * . .0.22 0.17 Leu 152 . A B . . . . 0.13 0.43 * . . −0.34 0.18 Leu 153 . A B. . . . −0.28 0.81 * . . −0.60 0.34 Cys 154 . A B . . . . −0.62 0.51 . *. −0.60 0.26 Gln 155 . A . . T . . −0.02 0.54 * * F −0.05 0.31 Ser 156 .. . . T T . −0.72 0.77 * . F 0.35 0.40 Ser 157 . . . . T T . −0.120.87 * . F 0.35 0.64 Gly 158 . . . . T T . 0.80 0.73 * . F 0.35 0.57 Trp159 . . . . T T . 1.26 0.33 * . F 0.65 0.84 Phe 160 . . . . . T C 0.940.37 * . F 0.45 0.97 Pro 161 . . . . . T C 0.66 0.47 * * F 0.30 1.41 Arg162 . . . . . T C 1.00 0.54 * * F 0.30 1.36 Pro 163 . . . . T T . 1.06−0.37 * * F 1.40 3.13 Thr 164 . . . . T . . 1.39 −0.24 * * F 1.20 2.13Ala 165 . . . . T . . 1.74 −0.67 * * F 1.50 2.17 Lys 166 . . . . T . .1.74 −0.24 . * F 1.20 1.39 Trp 167 . . . . T . . 1.63 −0.24 . * F 1.541.49 Lys 168 . . . . . . C 1.50 −0.33 . * F 1.68 2.55 Gly 169 . . . . .. C 1.81 −0.40 . * F 2.02 1.26 Pro 170 . . . . . T C 2.40 0.00 . * F1.96 2.08 Gln 171 . . . . T T . 1.54 −0.91 . * F 3.40 1.74 Gly 172 . . .. . T C 1.53 −0.23 . . F 2.56 1.45 Gln 173 . . B . . T . 1.18 −0.27 . .F 2.02 1.26 Asp 174 . . B . . . . 1.52 −0.21 . . F 1.82 1.05 Leu 175 . .B . . . . 1.43 −0.61 * * F 2.12 1.77 Ser 176 . . B . . T . 1.54−0.66 * * F 2.32 1.37 Thr 177 . . B . . T . 1.58 −1.06 * * F 2.66 1.60Asp 178 . . . . T T . 1.58 −0.57 * * F 3.40 2.80 Ser 179 . . . . . T C1.69 −0.86 * * F 2.86 3.37 Arg 180 . . . . T T . 2.50 −1.24 * * F 3.064.57 Thr 181 . . . . T T . 2.20 −1.73 * . F 3.06 4.57 Asn 182 . . . . TT . 2.48 −1.11 * . F 3.06 3.37 Arg 183 . . B . . T . 2.13 −1.00 * . F2.66 2.34 Asp 184 . . . . T T . 1.62 −0.57 * . F 3.40 1.61 Met 185 . . B. . T . 0.81 −0.37 * . . 2.06 0.82 His 186 . . B . . T . 1.12 0.01 * . .1.12 0.36 Gly 187 . . B . . T . 0.27 0.01 * * . 0.78 0.36 Leu 188 . . BB . . . 0.16 0.66 * * . −0.26 0.27 Phe 189 A . . B . . . −0.73 0.04 . *. −0.30 0.35 Asp 190 A . . B . . . −0.43 0.23 . * . −0.30 0.25 Val 191 A. . B . . . −1.21 0.19 . * . −0.30 0.40 Glu 192 A . . B . . . −1.18 0.19. * . −0.30 0.38 Ile 193 A . . B . . . −1.22 −0.11 . * . 0.30 0.33 Ser194 A . . B . . . −0.52 0.53 . * . −0.60 0.33 Leu 195 A A . B . . .−0.52 0.29 . * . −0.30 0.33 Thr 196 A A . B . . . 0.33 0.29 . * . −0.300.81 Val 197 A A . B . . . −0.26 0.00 . * . 0.55 0.98 Gln 198 A A . B .. . 0.29 0.11 . * F 0.50 1.20 Glu 199 A A . B . . . 0.29 −0.14 . . F1.20 0.82 Asn 200 . . . . T T . 0.21 −0.24 . . F 2.40 1.48 Ala 201 . . .. T T . 0.22 −0.20 . . F 2.50 0.60 Gly 202 . . . . T T . 0.41 −0.21 . .F 2.25 0.46 Ser 203 . . . . T T . 0.11 0.36 . . F 1.40 0.15 Ile 204 A .. . . . . −0.49 0.34 * * . 0.40 0.21 Ser 205 A . . . . . . −0.380.46 * * . −0.15 0.21 Cys 206 . . B . . . . 0.18 0.03 * * . −0.10 0.30Ser 207 . A B . . . . −0.07 0.14 * * . −0.30 0.58 Met 208 A A . . . . .0.20 −0.04 * * . 0.30 0.44 Arg 209 A A . . . . . 0.28 0.07 * * . −0.151.11 His 210 A A . . . . . 0.28 0.19 . . . −0.30 0.69 Ala 211 A A . . .. . 1.06 0.19 . . . −0.30 0.93 His 212 A A . . . . . 1.36 −0.43 * . .0.30 0.93 Leu 213 A A . . . . . 1.10 −0.43 * . . 0.45 1.18 Ser 214 A A .. . . . 0.99 −0.29 * . . 0.30 0.87 Arg 215 A A . . . . . 0.72 −0.79 * *F 0.90 1.10 Glu 216 A A . . . . . 1.42 −0.90 * * F 0.90 1.79 Val 217 A .. B . . . 0.60 −1.59 * * F 0.90 2.62 Glu 218 A . . B . . . 1.41−1.33 * * F 0.75 0.99 Ser 219 A . . B . . . 0.82 −0.93 * * F 0.75 0.99Arg 220 . . B B . . . 0.37 −0.24 * * F 0.45 0.94 Val 221 . . B B . . .0.37 −0.46 . * F 0.45 0.54 Gln 222 A . . B . . . 0.93 −0.46 . * . 0.640.67 Ile 223 A . . . . T . 1.04 0.07 * * . 0.78 0.36 Gly 224 A . . . . T. 1.46 0.07 * * F 1.27 0.95 Asp 225 . . . . T T . 1.39 −0.57 . * F 3.061.07 Trp 226 . . . . T T . 2.21 −0.97 . . F 3.40 3.06 Arg 227 . . B . .. . 1.87 −1.16 * . F 2.46 4.20 Arg 228 . . . . T T . 2.76 −1.16 * . F2.72 2.49 Lys 229 . . . . T T . 2.51 −0.76 * . F 2.38 4.10 His 230 . . .. T T . 2.17 −1.17 * . F 2.38 2.12 Gly 231 . . . . . T C 2.50 −0.74 * .F 2.18 1.07 Gln 232 . . . . T . . 2.50 −0.74 * . F 2.52 1.07 Ala 233 . .. . . . C 2.43 −0.74 * . F 2.66 1.54 Gly 234 . . . . T T . 2.14 −1.24 *. F 3.40 3.11 Lys 235 . . B . . T . 1.88 −0.91 * . F 2.66 2.81 Arg 236 .. . . T T . 1.92 −0.93 * . F 2.77 3.73 Lys 237 . . . . T T . 1.62−1.04 * . F 2.48 5.05 Tyr 238 . . B . . T . 2.18 −1.09 . . F 1.79 3.39Ser 239 . . B . . T . 1.63 −0.59 . . F 1.50 2.35 Ser 240 . . B . . T .1.34 0.10 . . F 0.50 0.82 Ser 241 . . B . . T . 1.23 0.86 . . F 0.150.82 His 242 . . B . . . . 0.89 0.10 * . . 0.20 1.03 Ile 243 . . B . . .. 0.43 0.10 * . . 0.15 1.03 Tyr 244 . . B . . . . 0.52 0.50 * . . −0.350.66 Asp 245 . . B . . . . 0.52 0.54 * . . −0.40 0.75 Ser 246 . . B . .. . 0.01 0.43 * . F −0.10 1.44 Phe 247 . . B . . T . −0.26 0.43 * . F−0.05 0.76 Pro 248 . . . . . T C −0.07 0.06 * . F 0.45 0.61 Ser 249 . .. . . T C −0.42 0.84 . . F 0.15 0.39 Leu 250 . . . . . T C −0.42 1.07 .. . 0.00 0.45 Ser 251 . . B . . . . −0.82 0.29 . . . −0.10 0.49 Phe 252. . B B . . . −0.37 0.64 . . . −0.60 0.31 Met 253 . . B B . . . −1.041.01 . . . −0.60 0.60 Asp 254 . . B B . . . −1.56 1.01 . . . −0.60 0.31Phe 255 . . B B . . . −0.63 1.31 . . . −0.60 0.30 Tyr 256 . . B B . . .−0.54 0.53 . . . −0.60 0.59 Ile 257 . . B B . . . −0.70 0.34 . . . −0.300.54 Leu 258 . . B B . . . −0.44 0.99 * . . −0.60 0.47 Arg 259 . . B B .. . −0.66 0.63 * . . −0.35 0.29 Pro 260 . . . B T . . −0.62 0.30 . * F0.75 0.65 Val 261 . . . B T . . −0.27 0.19 * * F 1.00 0.42 Gly 262 . . .. . T C 0.03 −0.50 * * F 2.35 0.42 Pro 263 . . . . T T . 0.89 0.00 * * F2.50 0.28 Cys 264 . . B . . T . −0.03 −0.43 * * F 1.85 0.74 Arg 265 . .B . . T . −0.68 −0.39 . * . 1.45 0.62 Ala 266 . A B . . . . −0.42 −0.17. * . 0.80 0.30 Lys 267 . A B . . . . −0.42 0.01 . * . −0.05 0.55 Leu268 . A B . . . . −0.52 −0.13 . * . 0.30 0.28 Val 269 . A B . . . .−0.67 0.36 . * . −0.30 0.40 Met 270 A A . . . . . −0.73 0.54 . * . −0.600.16 Gly 271 A A . . . . . −0.96 0.54 * * . −0.60 0.40 Thr 272 A A . . .. . −1.00 0.54 . * . −0.60 0.44 Leu 273 A A . . . . . −1.08 0.30 . * .−0.30 0.77 Lys 274 A A . . . . . −1.03 0.37 . * . −0.30 0.55 Leu 275 A A. . . . . −0.78 0.63 . * . −0.60 0.31 Gln 276 A A . . . . . −0.43 0.57. * . −0.60 0.37 Ile 277 . A B . . . . −0.98 −0.11 . * . 0.30 0.32 Leu278 A A . . . . . −0.20 0.53 . * . −0.60 0.29 Gly 279 A A . . . . .−0.94 0.34 . * . −0.30 0.23 Glu 280 A A . . . . . −0.99 0.73 . * . −0.600.28 Val 281 A A . . . . . −0.99 0.69 * * . −0.60 0.26 His 282 A A . . .. . −0.06 0.00 * * . 0.30 0.45 Phe 283 A A . . . . . 0.54 −0.43 . . .0.30 0.52 Val 284 A A . . . . . 0.86 0.00 . * . 0.45 1.07 Glu 285 A A .. . . . 0.56 −0.14 . . F 0.60 1.07 Lys 286 A . . . . T . 0.60 −0.26 * .F 1.00 1.66 Pro 287 A . . . . T . −0.18 −0.36 * . F 1.00 1.85 His 288 A. . . . T . 0.52 −0.31 * . F 0.85 0.88 Ser 289 A . . . . T . 0.49 0.09 *. . 0.10 0.76 Leu 290 A . . B . . . 0.19 0.77 . * . −0.60 0.35 Leu 291 .. B B . . . −0.20 0.73 . * . −0.60 0.34 Gln 292 . . B B . . . −0.33 0.66. . . −0.60 0.25 Ile 293 . . B B . . . −0.60 0.70 . . F −0.45 0.30 Ser294 . . B . . T . −0.61 0.40 . . F 0.25 0.49 Gly 295 . . . . T T . −0.110.20 . . F 0.65 0.41 Gly 296 . . . . T T . −0.11 0.29 . * F 0.65 0.84Ser 297 . . . . . T C −0.07 0.29 * . F 0.45 0.52 Thr 298 . . B B . . .0.87 −0.10 * . F 0.90 1.04 Thr 299 . . B B . . . 0.82 −0.53 * . F 1.502.11 Leu 300 . . B B . . . 0.96 −0.53 * . F 1.80 1.56 Lys 301 . . . B T. . 1.30 −0.49 * . F 2.20 1.67 Lys 302 . . . . T . . 1.39 −0.57 * . F3.00 1.86 Gly 303 . . . . . T C 1.41 −0.63 * . F 2.70 3.49 Pro 304 . . .. . T C 1.42 −0.40 * . F 2.10 1.83 Asn 305 . . . . . T C 1.53 −0.01 * .F 1.80 1.23 Pro 306 . . . . T T . 1.28 0.77 * . F 0.80 1.08 Trp 307 . .. . T . . 0.93 0.77 . . . 0.15 1.08 Ser 308 . . . . . . C 1.07 0.73 . .. −0.20 0.90 Phe 309 . . B . . . . 0.61 0.76 . . F −0.25 0.90 Pro 310 .. B . . . . 0.02 0.90 . . F −0.25 0.46 Ser 311 . . . . . T C −0.58 0.49. . F 0.15 0.34 Pro 312 . . . . T T . −0.99 0.79 . . F 0.35 0.33 Cys 313. . . . T T . −0.90 0.79 . . . 0.20 0.18 Ala 314 . . B . T T . −0.510.79 . . . 0.20 0.21 Leu 315 . . B . . . . −0.69 0.89 . . . −0.40 0.20Phe 316 . . B . . . . −0.78 0.89 . . . −0.40 0.47 Pro 317 . . B . . . .−0.96 0.74 . . . −0.40 0.60 Thr 318 . . B . . . . −0.68 0.67 . . . −0.400.93

TABLE IX Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . B. . . . −0.69 0.51 . . . −0.40 0.26 Ala 2 . . B . . . . −0.64 0.51 . . .−0.40 0.31 Gly 3 . . B . . . . −1.07 0.51 . . . −0.40 0.24 Ile 4 . . B .. . . −1.49 0.77 . . . −0.40 0.20 Pro 5 . . B . . . . −1.80 0.84 . . F−0.25 0.17 Gly 6 . . B B . . . −2.01 1.13 . . . −0.60 0.14 Leu 7 . . B B. . . −2.23 1.39 . . . −0.60 0.17 Leu 8 . . B B . . . −2.59 1.39 . . .−0.60 0.09 Phe 9 . . B B . . . −2.40 1.74 . . . −0.60 0.08 Leu 10 . . BB . . . −3.00 2.10 . . . −0.60 0.08 Leu 11 . . B B . . . −3.47 2.10 . .. −0.60 0.08 Phe 12 . . B B . . . −3.32 2.10 . . . −0.60 0.08 Phe 13 . .B B . . . −3.10 1.89 . . . −0.60 0.05 Leu 14 . . B B . . . −3.26 1.70 .. . −0.60 0.06 Leu 15 . . B B . . . −2.79 1.66 . . . −0.60 0.05 Cys 16 .. B B . . . −1.98 1.30 * . . −0.60 0.06 Ala 17 . . B B T . . −2.13 0.91. . . −0.20 0.13 Val 18 . . B B . . . −1.73 0.87 . . . −0.60 0.12 Gly 19. . B B . . . −1.13 0.57 * . . −0.60 0.29 Gln 20 . . B B . . . −0.570.43 . . F −0.45 0.45 Val 21 . . B B . . . −0.20 0.69 . . F −0.45 0.95Ser 22 . . B . . T . −0.20 0.43 . . F 0.10 1.28 Pro 23 . . B . . T .0.44 0.50 . . F −0.05 0.75 Tyr 24 . . . . T T . 0.50 0.53 . * . 0.351.56 Ser 25 . . . . . T C 0.54 0.80 . * . 0.15 1.22 Ala 26 . . . . . . C1.19 0.41 . . . −0.05 1.58 Pro 27 . . . . T . . 1.18 0.41 . * . 0.151.56 Trp 28 . . . . T . . 1.10 0.14 . * . 0.45 1.68 Lys 29 . . . . . T C1.13 0.67 . * F 0.30 1.75 Pro 30 . . . . T T . 0.84 0.60 . * F 0.50 1.75Thr 31 . . . . T T . 1.19 0.67 * * F 0.50 1.68 Trp 32 . . B . . T . 1.510.51 * * . −0.05 1.32 Pro 33 . . B . . T . 0.99 0.51 * * . −0.05 1.67Ala 34 . . . . T T . 0.73 0.77 * * . 0.20 0.95 Tyr 35 . . . . T T . 0.090.71 . . . 0.35 1.40 Arg 36 . . B . . T . −0.46 0.44 . * . −0.20 0.67Leu 37 . . B B . . . −0.98 0.66 . * . −0.60 0.49 Pro 38 . . B B . . .−0.98 0.84 * * . −0.60 0.26 Val 39 . . B B . . . −0.39 0.51 * * . −0.600.21 Val 40 . . B B . . . −0.44 0.91 * * . −0.60 0.43 Leu 41 . . B B . .. −0.87 0.61 * * F −0.45 0.37 Pro 42 . . B . . T . −0.87 0.67 . . F−0.05 0.73 Gln 43 . . B . . T . −0.66 0.71 . * F −0.05 0.81 Ser 44 . . B. . T . −0.61 0.47 . . F 0.10 1.57 Thr 45 . . B . . T . −0.34 0.47 * . F−0.05 0.84 Leu 46 . . B . . . . 0.51 0.54 * . F −0.25 0.49 Asn 47 . . B. . . . 0.51 0.14 . . . 0.14 0.73 Leu 48 . . B . . . . 0.51 0.19 * * .0.38 0.78 Ala 49 . . B . . . . 0.11 −0.30 . . . 1.37 1.59 Lys 50 . . . .. T C 0.08 −0.20 . . F 2.01 0.85 Pro 51 . . . . . T C 0.30 −0.17 . * F2.40 1.02 Asp 52 . . . . . T C 0.30 −0.36 . * F 2.16 1.02 Phe 53 . . B .. T . 0.52 −0.86 . * . 1.72 0.89 Gly 54 A A . . . . . 1.16 −0.36 . * .0.78 0.58 Ala 55 A A . . . . . 0.30 −0.79 . * . 0.84 0.69 Glu 56 A A . .. . . 0.51 −0.10 . * . 0.30 0.66 Ala 57 A A . . . . . −0.34 −0.89 . * F0.90 1.16 Lys 58 A A . . . . . 0.06 −0.67 . * F 0.75 0.85 Leu 59 . A B .. . . 0.10 −0.79 . * . 0.60 0.66 Glu 60 . A B . . . . 0.39 −0.40 . * .0.30 0.87 Val 61 . A B . . . . −0.28 −0.51 . * F 1.00 0.58 Ser 62 . A .. T . . −0.03 0.06 . * F 0.75 0.38 Ser 63 . . . . T T . −0.29 −0.20 . *F 2.00 0.22 Ser 64 . . . . T T . 0.52 0.23 . * F 1.65 0.45 Cys 65 . . .. T T . −0.14 −0.01 . . F 2.50 0.58 Gly 66 . . . . . T C 0.68 0.17 . . F1.45 0.23 Pro 67 . . . . T . . 1.02 0.29 . . F 1.45 0.24 Gln 68 . . . .T . . 0.98 −0.10 . . . 1.90 0.89 Cys 69 . . B . . . . 0.97 −0.24 . . .1.50 0.89 His 70 . . . . T T . 1.42 −0.19 . . F 2.25 0.83 Lys 71 . . . .T T . 0.96 −0.19 . . F 2.50 0.74 Gly 72 . . . . T T . 0.96 0.10 . . F1.80 1.14 Thr 73 . . . . . T C 0.64 −0.04 . . F 1.95 1.29 Pro 74 . . . .. . C 1.07 −0.06 . . F 1.35 0.93 Leu 75 . . . . . . C 1.10 0.70 . . F0.61 1.48 Pro 76 . . . . . T C 1.06 0.27 . . F 1.12 1.77 Thr 77 . . B .. T . 0.81 −0.21 . . F 1.78 1.98 Tyr 78 . . B . . T . 1.17 −0.14 . . F2.04 2.43 Glu 79 . . B . . T . 1.38 −0.83 . . F 2.60 3.14 Glu 80 . A B .. . . 1.94 −0.86 * . F 1.94 3.77 Ala 81 A A . . . . . 1.34 −0.59 * . F1.68 3.77 Lys 82 . A B . . . . 1.36 −0.66 * . F 1.42 1.79 Gln 83 . A B .. . . 1.36 −0.27 * . F 0.86 1.39 Tyr 84 . A B . . . . 1.36 0.49 . . .−0.45 2.15 Leu 85 . . B B . . . 1.04 −0.01 . * . 0.45 1.87 Ser 86 . . BB . . . 0.82 0.47 . . . −0.45 1.55 Tyr 87 . A B B . . . 0.53 0.76 . . .−0.60 0.82 Glu 88 . A B B . . . −0.06 0.76 . . . −0.45 1.55 Thr 89 . A BB . . . 0.19 0.57 . . . −0.45 1.17 Leu 90 . A B B . . . 0.66 0.59 . . .−0.45 1.20 Tyr 91 . . B . . T . 0.66 0.26 * . . 0.36 0.69 Ala 92 . . . .. T C 1.01 0.64 * . . 0.52 0.64 Asn 93 . . . . . T C 0.70 0.16 * . F1.38 1.52 Gly 94 . . . . . T C 1.01 −0.04 * . F 2.24 1.40 Ser 95 . . . .. . C 1.51 −0.80 * . F 2.60 2.39 Arg 96 . . . B . . C 1.76 −0.81 * * F2.14 2.15 Thr 97 . . B B . . . 1.49 −0.81 * * F 1.68 3.76 Glu 98 . . B B. . . 1.14 −0.60 * * F 1.42 2.08 Thr 99 . . B B . . . 0.60 −0.56 . * F1.16 1.05 Gln 100 . . B B . . . 0.66 0.13 * * F −0.15 0.51 Val 101 . . BB . . . −0.34 0.40 * * . −0.60 0.46 Gly 102 . . B B . . . −0.84 1.09 . *. −0.60 0.22 Ile 103 . . B B . . . −1.14 1.29 . * . −0.60 0.11 Tyr 104 .. B B . . . −1.13 1.27 . . . −0.60 0.19 Ile 105 . . B B . . . −1.43 1.01. . . −0.60 0.26 Leu 106 . . B B . . . −0.92 0.97 . . . −0.35 0.50 Ser107 . . B . . T . −0.58 0.71 . . F 0.45 0.32 Ser 108 . . . . . T C −0.03−0.04 * . F 1.80 0.75 Ser 109 . . . . . T C −0.38 −0.30 * . F 2.05 0.90Gly 110 . . . . T T . 0.51 −0.49 * . F 2.50 0.68 Asp 111 . A . . T . .1.29 −0.47 . * F 1.85 0.88 Gly 112 . A . . . . C 1.70 −0.36 . . F 1.400.89 Ala 113 . A B . . . . 2.00 −0.74 . . F 1.40 1.77 Gln 114 . A B . .. . 2.00 −1.17 . . . 1.34 1.77 His 115 . A B . . . . 2.00 −0.79 . . F1.58 2.39 Arg 116 . A B . . . . 1.70 −0.79 . . F 1.92 2.34 Asp 117 . . .. T T . 1.74 −0.90 . . F 3.06 1.81 Ser 118 . . . . T T . 1.99 −0.91 . *F 3.40 1.79 Gly 119 . . . . T T . 2.03 −0.99 * . F 2.91 0.90 Ser 120 . .. . T T . 1.77 −0.99 . * F 3.02 1.08 Ser 121 . . . . . . C 1.77 −0.60 .. F 2.58 1.08 Gly 122 . . . . . T C 1.88 −0.99 . * F 2.74 2.14 Lys 123 .. . . T T . 2.22 −1.41 . . F 2.90 3.13 Ser 124 . . . . . T C 2.68 −1.80. . F 3.00 4.66 Arg 125 . . B . . T . 2.98 −2.19 . . F 2.50 9.23 Arg 126. . B . . . . 2.39 −2.21 . . F 2.00 7.99 Lys 127 . . B B . . . 2.49−1.53 . . F 1.50 4.18 Arg 128 . . B B . . . 2.10 −1.16 . . F 1.20 3.35Gln 129 . . B B . . . 2.16 −0.73 . . . 0.75 1.69 Ile 130 . . B B . . .2.04 0.03 . . . −0.15 1.32 Tyr 131 . . B B . . . 1.63 0.03 . . . −0.151.13 Gly 132 . . B . . . . 1.70 0.41 * * . −0.40 0.87 Tyr 133 . . B . .. . 0.89 0.01 * * . 0.05 2.44 Asp 134 . . B . . T . 0.59 0.11 . * F 0.401.35 Ser 135 . . B . . T . 0.59 −0.26 . * F 1.00 1.83 Arg 136 . . B . .T . 0.13 0.00 . * F 0.25 0.82 Phe 137 . . B . . T . 0.13 0.03 . * . 0.100.42 Ser 138 . . B B . . . 0.42 0.46 . * . −0.47 0.31 Ile 139 . . B B .. . 0.42 0.07 * * . −0.04 0.32 Phe 140 . . B B . . . 0.02 0.07 * * .0.09 0.62 Gly 141 . . . . T T . −0.90 0.07 * * . 1.02 0.40 Lys 142 . . .. T T . −1.01 0.37 . . F 1.30 0.47 Asp 143 . . . . T T . −0.71 0.37 . .F 1.17 0.45 Phe 144 . . B . . T . −0.07 −0.01 . . . 1.09 0.73 Leu 145 .. B . . . . 0.42 0.31 . . . 0.16 0.57 Leu 146 . . B . . . . 0.07 0.74 .. . −0.27 0.53 Asn 147 . . B . . . . −0.28 1.53 . . . −0.40 0.53 Tyr 148. . . . . T C −0.59 1.13 . * . 0.00 0.85 Pro 149 . . . . T T . −0.190.93 . * . 0.35 1.50 Phe 150 . . . . T T . −0.23 0.63 * * . 0.35 1.25Ser 151 . . B . . T . 0.62 0.87 * * F −0.05 0.59 Thr 152 . . B B . . .−0.19 0.11 . * F −0.15 0.76 Ser 153 . . B B . . . −0.24 0.37 . * F −0.150.73 Val 154 . . B B . . . −0.34 −0.03 . * F 0.45 0.73 Lys 155 . . B B .. . 0.01 0.07 . * F −0.15 0.73 Leu 156 . . B B . . . −0.36 0.01 . * F−0.15 0.54 Ser 157 . . B . . T . −0.36 0.20 . * F 0.25 0.39 Thr 158 . .B . . T . −0.40 0.04 . * F 0.25 0.28 Gly 159 . . . . T T . 0.14 0.47 . *F 0.35 0.34 Cys 160 . . . . T T . −0.71 0.27 . . F 0.65 0.36 Thr 161 . .B . . . . −0.76 0.57 . . F −0.25 0.21 Gly 162 . . B . . . . −1.04 0.73 .. F −0.25 0.16 Thr 163 . A B . . . . −0.73 0.80 . . F −0.45 0.29 Leu 164. A B . . . . −0.34 0.23 . . . −0.30 0.35 Val 165 . A B . . . . 0.29−0.26 . . . 0.30 0.71 Ala 166 . A B . . . . −0.26 −0.19 . . . 0.30 0.67Glu 167 A A . . . . . −0.72 −0.03 . . . 0.30 0.60 Lys 168 A A . . . . .−0.72 −0.03 . . . 0.30 0.67 His 169 A A . . . . . −0.50 −0.19 . . . 0.300.95 Val 170 A A . . . . . −0.23 −0.19 * . . 0.30 0.56 Leu 171 A A . . .. . 0.32 0.31 * . . −0.30 0.28 Thr 172 A A . . . . . −0.34 0.81 * . .−0.60 0.28 Ala 173 A A . . . . . −1.28 0.89 * . . −0.60 0.20 Ala 174 A A. . . . . −1.28 0.93 * . . −0.60 0.17 His 175 . A B . . . . −0.42 0.74 *. . −0.32 0.16 Cys 176 . A B . . . . 0.04 0.26 . . . 0.26 0.27 Ile 177 .A B . . . . 0.40 0.19 * . . 0.54 0.26 His 178 . . . . T T . 0.68 −0.31 *. . 2.22 0.39 Asp 179 . . . . T T . 1.02 −0.33 * . F 2.80 1.04 Gly 180 .. . . T T . 0.20 −0.14 * * F 2.52 2.33 Lys 181 . . . . T T . 0.91−0.19 * * F 2.24 1.27 Thr 182 . . B B . . . 1.46 −0.69 * * F 1.46 1.52Tyr 183 . . B B . . . 1.18 −0.26 * * F 0.88 1.52 Val 184 . . B B . . .1.18 −0.20 * * F 0.60 1.10 Lys 185 . . B B . . . 1.57 0.20 * * F 0.121.32 Gly 186 . . B . . . . 0.71 −0.29 * * F 1.04 1.68 Thr 187 . . B . .. . 1.13 −0.36 . * F 1.16 1.87 Gln 188 . . B . . . . 0.52 −1.00 * * F1.58 1.83 Lys 189 . . B B . . . 1.03 −0.36 * * F 1.20 1.37 Leu 190 . . BB . . . 0.29 −0.36 * * F 0.93 0.94 Arg 191 . . B B . . . −0.18 −0.06 * *. 0.66 0.47 Val 192 . . B B . . . 0.18 0.23 . * . −0.06 0.19 Gly 193 . .B B . . . −0.03 0.23 . * . −0.18 0.47 Phe 194 . . B B . . . −0.03 −0.03. * . 0.64 0.37 Leu 195 . . B B . . . 0.08 −0.03 . * . 1.13 1.00 Lys 196. . B . . T . 0.01 0.11 . * F 1.27 0.88 Pro 197 . . B . . T . 0.87−0.31 * * F 2.36 2.02 Lys 198 . . . . T T . 0.87 −1.10 * * F 3.40 4.10Phe 199 . . B . . T . 1.22 −1.36 * * F 2.66 2.03 Lys 200 . . B . . . .2.14 −0.93 * * F 2.12 1.30 Asp 201 . . . . T T . 1.76 −1.36 * * F 2.381.27 Gly 202 . . . . T T . 1.38 −0.93 * * F 2.04 1.45 Gly 203 . . . . TT . 1.33 −1.21 * * F 1.85 0.73 Arg 204 . . . . . T C 2.03 −0.81 * * F1.95 0.71 Gly 205 . . . . . . C 1.69 −0.81 * . F 2.20 1.19 Ala 206 . . .. . . C 1.38 −0.86 * . F 2.50 1.62 Asn 207 . . . . . T C 1.42 −0.80 * .F 3.00 1.19 Asp 208 . . . . . T C 1.18 −0.41 * . F 2.40 1.61 Ser 209 . .. . . T C 0.47 −0.34 * . F 2.10 1.61 Thr 210 . . . . . T C 0.60 −0.23 *. F 1.65 0.99 Ser 211 . . . . . . C 1.19 −0.20 . . F 1.15 0.92 Ala 212 .A . . . . C 1.19 −0.20 . . F 0.80 1.19 Met 213 . A B . . . . 0.59−0.19 * . . 0.45 1.42 Pro 214 . A . . . . C 0.93 −0.06 . * F 0.80 1.05Glu 215 A A . . . . . 0.54 −0.44 . * F 0.60 2.08 Gln 216 A A . . . . .0.84 −0.16 . * F 0.60 1.82 Met 217 A A . . . . . 1.14 −0.37 . * F 0.602.04 Lys 218 A . . B . . . 0.86 0.11 * * . −0.15 1.24 Phe 219 A . . B .. . 1.18 0.80 * * . −0.60 0.50 Gln 220 . . B B . . . 0.32 0.40 . * .−0.60 0.99 Trp 221 . . B B . . . 0.37 0.43 * * . −0.60 0.37 Ile 222 . .B B . . . 1.08 0.43 * . . −0.37 0.85 Arg 223 . . B B . . . 0.72 −0.36 .. . 0.76 0.96 Val 224 . . B B . . . 1.39 −0.27 * . . 1.14 1.32 Lys 225 .. . B T . . 0.53 −0.69 * . F 2.22 2.56 Arg 226 . . . B T . . 0.61−0.73 * . F 2.30 0.97 Thr 227 . . . B T . . 1.54 −0.30 * * F 1.92 2.02His 228 . . . B . . C 1.09 −0.94 * . F 1.79 2.02 Val 229 . . . B . . C1.66 −0.51 . . F 1.56 1.02 Pro 230 . . B . . T . 0.72 0.40 . . F 0.180.74 Lys 231 . . . . T T . 0.66 0.60 . * F 0.35 0.38 Gly 232 . . . . T T. 0.62 0.10 * * F 0.80 1.03 Trp 233 . . B . . T . 0.66 −0.11 * * F 0.850.66 Ile 234 . . B . . . . 0.92 −0.14 * * F 0.92 0.53 Lys 235 . . B . .. . 1.13 0.36 . * F 0.59 0.54 Gly 236 . . . . . . C 1.09 0.33 * * F 1.060.83 Asn 237 . . . . . T C 0.54 −0.59 * * F 2.58 1.98 Ala 238 . . . . .T C 0.49 −0.59 * * F 2.70 0.69 Asn 239 . . . . . T C 0.78 −0.16 * * F2.13 0.69 Asp 240 . . . . T T . 0.73 0.03 . * F 1.46 0.43 Ile 241 . . B. . . . 0.83 −0.37 . * . 1.04 0.71 Gly 242 . . B . . . . 0.83 −0.11 . *. 0.77 0.69 Met 243 . . B . . . . 1.18 −0.51 * . . 0.80 0.69 Asp 244 . .B . . T . 0.59 0.24 * * . 0.25 1.54 Tyr 245 . . B . . T . −0.22 0.06 . *. 0.25 1.57 Asp 246 . . B . . T . −0.14 0.31 . * . 0.25 1.31 Tyr 247 . .B . . T . 0.20 0.39 . . . 0.10 0.65 Ala 248 A A . . . . . −0.01 0.39 . *. −0.30 0.71 Leu 249 A A . . . . . 0.03 0.31 * . . −0.30 0.35 Leu 250 AA . . . . . 0.32 0.31 * . . −0.30 0.45 Glu 251 A A . . . . . 0.11−0.44 * . . 0.30 0.89 Leu 252 A A . . . . . 0.32 −0.51 * . F 0.90 1.67Lys 253 A A . . . . . 0.96 −0.70 * . F 0.90 2.76 Lys 254 A . . . . T .1.88 −1.39 * . F 1.30 3.18 Pro 255 A . . . . T . 2.73 −1.39 * . F 1.307.56 His 256 A . . . . T . 2.03 −2.07 * . F 1.30 7.56 Lys 257 A . . . .T . 2.24 −1.29 * . F 1.30 3.27 Arg 258 A . . . . . . 2.24 −0.67 * . F1.10 2.09 Lys 259 . . B . . . . 1.31 −1.10 * . F 1.10 3.08 Phe 260 . . BB . . . 1.18 −0.91 * . . 0.75 1.08 Met 261 . . B B . . . 0.36 −0.49 * *. 0.30 0.55 Lys 262 . . B B . . . 0.01 0.16 * * . −0.30 0.20 Ile 263 . .B B . . . −0.31 0.54 * * . −0.60 0.31 Gly 264 . . B B . . . −0.570.19 * * . −0.02 0.49 Val 265 . . . B . . C −0.46 0.00 * * F 0.61 0.38Ser 266 . . . B . . C 0.19 0.50 . * F 0.59 0.55 Pro 267 . . . . . T C0.14 −0.19 . * F 2.32 1.10 Pro 268 . . . . T T . 0.22 −0.21 . . F 2.802.57 Ala 269 . . . . T T . 0.36 −0.17 * . F 2.52 1.58 Lys 270 . . B . .T . 0.87 −0.13 * . F 1.84 1.58 Gln 271 . . B . . . . 0.82 −0.13 . . F1.36 1.01 Leu 272 . . B . . T . 1.14 −0.13 . * F 1.13 0.99 Pro 273 . . B. . T . 0.47 −0.63 . * F 1.15 0.97 Gly 274 . . . . T T . 1.02 0.06 . * F0.65 0.39 Gly 275 . . B . . T . 0.28 0.16 . * F 0.25 0.65 Arg 276 . . BB . . . −0.02 0.26 . * F −0.15 0.36 Ile 277 . . B B . . . 0.44 0.21 . *. −0.14 0.49 His 278 . . B B . . . 0.41 0.21 * * . 0.02 0.49 Phe 279 . .B . . T . 0.76 0.54 * * . 0.28 0.39 Ser 280 . . B . . T . 1.10 0.54 . *. 0.44 0.94 Gly 281 . . . . T T . 0.99 0.26 * * F 1.60 1.11 Tyr 282 . .. . T T . 1.99 −0.24 . * F 2.04 2.14 Asp 283 . . . . T . . 1.81 −1.03 .. F 2.32 3.12 Asn 284 . . . . T . . 2.17 −0.99 . . F 2.50 4.88 Asp 285 .. . . T . . 2.47 −0.99 . . F 2.68 3.08 Arg 286 . . . . . T C 2.00−1.34 * . F 2.86 2.97 Pro 287 . . . . T T . 1.39 −0.66 * . F 3.40 1.52Gly 288 . . . . T T . 1.14 −0.41 * . F 2.61 0.68 Asn 289 . . B . . T .1.26 0.34 * * F 1.27 0.54 Leu 290 . . B B . . . 0.56 0.34 * * . 0.380.69 Val 291 . . B B . . . −0.22 0.70 * . . −0.26 0.60 Tyr 292 . . B B .. . −0.01 0.84 * * . −0.60 0.20 Arg 293 . A B B . . . −0.52 0.44 * * .−0.60 0.40 Phe 294 . A B B . . . −0.48 0.40 * . . −0.60 0.40 Cys 295 . AB B . . . 0.33 −0.24 * . . 0.30 0.52 Asp 296 . A B B . . . 1.19 −1.00 *. . 0.60 0.44 Val 297 . A B . . . . 1.12 −1.00 * . . 0.60 0.88 Lys 298 .A . . T . . 0.77 −1.30 * . F 1.30 2.37 Asp 299 . A . . T . . 1.47−1.11 * . F 1.30 2.23 Glu 300 . A B . T . . 1.32 −1.11 * . F 1.30 5.01Thr 301 . A . B T . . 0.51 −1.07 * . F 1.30 2.06 Tyr 302 . A . B T . .1.12 −0.39 * . . 0.85 1.02 Asp 303 . A B B . . . 1.08 0.37 . . . −0.300.92 Leu 304 . A B B . . . 1.08 0.77 * . . −0.45 1.11 Leu 305 . A B B .. . 0.41 0.69 * . . −0.45 1.22 Tyr 306 . A B B . . . 0.72 0.50 * . .−0.60 0.39 Gln 307 . A B B . . . 0.67 0.50 . . . −0.60 0.80 Gln 308 . AB B . . . 0.67 0.20 . . F 0.00 1.29 Cys 309 . A B B . . . 1.27 −0.09 . .F 0.60 1.43 Asp 310 . . . B T . . 1.73 −0.41 . . F 1.25 1.28 Ser 311 . .B . . . . 1.39 −0.39 . . F 1.15 0.73 Gln 312 . . B . . T . 1.09 −0.29 .. F 1.75 1.37 Pro 313 . . . . . T C 0.74 −0.47 . * F 2.20 1.10 Gly 314 .. . . T T . 1.11 −0.04 . . F 2.50 0.81 Ala 315 . . . . T T C 0.77 −0.04. . F 2.25 0.63 Ser 316 . . . . . T C 0.21 −0.01 . . F 1.80 0.40 Gly 317. . . . . T C −0.03 0.20 . . F 0.95 0.30 Ser 318 . . B . . T . −0.680.53 . * F 0.20 0.47 Gly 319 . . B . . T . −0.22 0.67 . * F −0.05 0.26Val 320 . . B B . . . −0.23 0.29 * * . −0.30 0.51 Tyr 321 . . B B . . .−0.22 0.47 * . . −0.60 0.38 Val 322 . . B B . . . 0.17 1.00 * * . −0.600.40 Arg 323 . . B B . . . 0.58 0.57 * * . −0.45 1.09 Met 324 . . B B .. . 0.92 −0.07 * * . 0.45 1.36 Trp 325 . . B B . . . 1.74 −0.43 * * .0.45 3.17 Lys 326 . . B B . . . 1.99 −0.57 * * . 0.75 2.20 Arg 327 . A .. T . . 2.89 −0.17 * * F 1.00 3.85 Gln 328 . A . . . . C 2.49 −0.79 * *F 1.10 7.33 His 329 . A . . . . C 3.09 −0.79 * . F 1.10 3.85 Gln 330 . A. . . . C 3.49 −0.79 * . F 1.10 3.41 Lys 331 . A . . T . . 3.49 −0.79 *. F 1.30 3.85 Trp 332 . A . . T . . 2.49 −1.19 * * F 1.30 5.66 Glu 333 .A . . . . C 1.60 −1.00 * . F 1.10 2.29 Arg 334 . A B B . . . 1.29−0.71 * . F 0.75 0.80 Lys 335 . A B B . . . 0.69 −0.29 * . . 0.30 0.76Ile 336 . A B B . . . −0.24 −0.59 * . . 0.60 0.43 Ile 337 . A B B . . .−0.26 0.10 * . . −0.30 0.15 Gly 338 . A B B . . . −0.60 0.49 * . . −0.600.10 Met 339 . . B B . . . −0.74 0.91 * . . −0.60 0.15 Ile 340 . . B B .. . −0.79 0.73 . . . −0.60 0.28 Ser 341 . . . . . T C −0.19 0.44 . . .0.00 0.50 Gly 342 . . . . . T C −0.16 0.93 . . . 0.00 0.53 His 343 . . .. . T C 0.19 0.96 . . . 0.00 0.56 Gln 344 . . B . . T . 0.19 0.27 . * .0.10 0.70 Trp 345 . . B . . . . 1.08 0.50 . * . −0.40 0.70 Val 346 . . B. . . . 1.03 0.07 . * . 0.20 0.86 Asp 347 . . B . . T . 1.08 0.00 . * .0.70 0.49 Met 348 . . . . T T . 0.90 −0.01 . * F 2.15 0.62 Asp 349 . . .. T T . 0.90 −0.50 . * F 2.60 1.30 Gly 350 . . . . . T C 1.19 −0.74 . *F 3.00 1.35 Ser 351 . . . . . T C 1.34 −0.74 . * F 2.70 2.35 Pro 352 . .. . . T C 1.03 −0.57 * * F 2.40 1.22 Gln 353 . . . . T T . 1.74 −0.09 *. F 2.25 1.78 Glu 354 . . B . . T . 1.40 −0.51 * . F 2.10 2.60 Phe 355 .. B . . . . 1.08 −0.47 * . F 1.55 1.67 Thr 356 . . . . T T . 1.08−0.33 * . F 2.25 0.52 Arg 357 . . . . T T . 1.29 −0.34 * * F 2.50 0.40Gly 358 . . . . T T . 0.40 −0.34 * * F 2.25 0.80 Cys 359 . . . . T T .0.09 −0.44 * * F 2.00 0.39 Ser 360 . . . B . . C 0.58 −0.44 * . F 1.150.29 Glu 361 . . . B T . . 0.08 −0.01 * * F 1.10 0.45 Ile 362 . . B B .. . −0.03 0.24 * * F −0.15 0.69 Thr 363 . . B B . . . 0.07 0.07 . . F−0.15 0.89 Pro 364 . . B B . . . −0.16 0.44 . . F −0.45 0.80 Leu 365 . .B B . . . −0.07 1.13 * . . −0.60 0.80 Gln 366 . . B B . . . −0.07 0.87 *. . −0.60 0.86 Tyr 367 . . B B . . . −0.07 0.39 * . . −0.30 0.93 Ile 368. . B B . . . −0.06 0.64 * * . −0.60 0.79 Pro 369 . . B B . . . −0.730.34 . * . −0.30 0.61 Asp 370 . . B B . . . −0.27 0.63 * * F −0.45 0.27Ile 371 . . B B . . . −1.12 0.30 * * F −0.15 0.39 Ser 372 . . B B . . .−1.27 0.26 . * . −0.30 0.19 Ile 373 . . B B . . . −0.77 0.26 . * . −0.300.14 Gly 374 . . B B . . . −0.94 0.69 . * . −0.60 0.26 Val 375 . . B B .. . −1.33 0.43 . * . −0.60 0.25

TABLE X Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . B B. . . −1.26 0.43 . . . −0.60 0.37 Ala 2 . . B B . . . −1.68 0.64 . . .−0.60 0.22 Ser 3 . . B B . . . −1.50 0.90 . . . −0.60 0.14 Val 4 . . B B. . . −1.41 0.90 . . . −0.60 0.22 Val 5 . . B B . . . −1.37 0.67 . . .−0.60 0.29 Leu 6 . . B . . T . −1.07 0.60 . . F −0.05 0.21 Pro 7 . . . .T T . −0.48 0.60 . . F 0.35 0.39 Ser 8 . . . . T T . −0.84 0.36 . . F0.65 0.90 Gly 9 . . . . T T . −0.58 0.29 . . F 0.65 0.58 Ser 10 . . . .. T C −0.31 0.10 . . F 0.45 0.38 Gln 11 A . . . . T . −0.09 0.17 . . F0.25 0.29 Cys 12 A . . . . T . −0.47 0.29 . . . 0.10 0.29 Ala 13 A . . .. T . −0.76 0.36 . . . 0.10 0.22 Ala 14 A A . . . . . −1.00 0.47 . . .−0.60 0.13 Ala 15 A A . . . . . −1.29 0.57 . . . −0.60 0.24 Ala 16 A A .. . . . −1.88 0.50 . . . −0.60 0.24 Ala 17 A A . . . . . −1.42 0.50 . .. −0.60 0.24 Ala 18 A A . . . . . −1.04 0.43 . . . −0.60 0.37 Ala 19 A A. . . . . −0.80 0.36 . . . −0.30 0.57 Ala 20 A A . . . . . −1.020.29 * * . −0.30 0.56 Pro 21 A . . . . T . −0.32 0.47 * * F −0.05 0.46Pro 22 A . . . . T . −0.54 −0.03 . * F 0.85 0.89 Gly 23 A . . . . T .0.16 0.16 . * F 0.25 0.72 Leu 24 A . . . . T . −0.07 −0.34 . * . 0.700.92 Arg 25 . A B . . . . −0.29 −0.09 . * . 0.30 0.49 Leu 26 . A B . . .. −0.89 0.17 . * . −0.30 0.41 Arg 27 . A B . . . . −1.49 0.43 . * .−0.60 0.41 Leu 28 . A B . . . . −1.96 0.43 . * . −0.60 0.17 Leu 29 . A B. . . . −1.84 1.11 . * . −0.60 0.17 Leu 30 . A B . . . . −2.26 1.21 . *. −0.60 0.08 Leu 31 A A . . . . . −2.03 1.60 * * . −0.60 0.12 Leu 32 A A. . . . . −2.73 1.41 * * . −0.60 0.15 Phe 33 A A . . . . . −2.51 1.23 .. . −0.60 0.18 Ser 34 A A . . . . . −2.51 1.04 . . . −0.60 0.23 Ala 35 AA . . . . . −2.59 1.04 . . . −0.60 0.23 Ala 36 A A . . . . . −1.99 1.04. . . −0.60 0.18 Ala 37 A A . . . . . −1.49 0.69 . . . −0.60 0.21 Leu 38. A B . . . . −1.13 0.79 . . . −0.60 0.30 Ile 39 . A B . . . . −0.830.71 . . . −0.32 0.30 Pro 40 . A B . . . . −0.59 0.21 . . F 0.41 0.49Thr 41 . . . . T . . 0.00 0.14 . . F 1.29 0.59 Gly 42 . . . . T T . 0.59−0.14 . . F 2.52 1.45 Asp 43 . . . . T T . 0.59 −0.43 . . F 2.80 1.51Gly 44 . . . . . T C 0.78 −0.17 . . F 2.17 0.86 Gln 45 . . B . . T .0.68 0.13 * . F 1.09 0.75 Asn 46 . . B B . . . 1.03 0.19 * . F 0.41 0.65Leu 47 . . B B . . . 1.38 0.19 * . F 0.28 1.32 Phe 48 . . B B . . . 0.52−0.24 * . F 0.60 1.27 Thr 49 . . B B . . . 0.56 0.00 * . F −0.15 0.59Lys 50 . . B B . . . −0.30 0.09 * . F 0.00 1.02 Asp 51 . . B B . . .−1.19 0.04 * . F −0.15 0.88 Val 52 . . B B . . . −0.38 −0.06 * . . 0.300.43 Thr 53 . . B B . . . −0.02 −0.54 * . . 0.60 0.37 Val 54 . . B B . .. 0.29 −0.11 * . . 0.30 0.22 Ile 55 A . . B . . . −0.61 −0.11 * . . 0.300.51 Glu 56 A . . B . . . −1.20 −0.11 . . F 0.45 0.26 Gly 57 A . . . . .. −0.66 −0.10 * . F 0.65 0.36 Glu 58 A . . B . . . −1.23 −0.26 . . F0.45 0.74 Val 59 A . . B . . . −0.68 −0.26 . . . 0.30 0.30 Ala 60 A . .B . . . −0.46 0.13 . * . −0.30 0.40 Thr 61 A . . B . . . −0.46 0.27 . *. −0.30 0.12 Ile 62 A . . B . . . −0.97 0.67 . * . −0.60 0.29 Ser 63 A .. B . . . −0.97 0.67 * . . −0.60 0.21 Cys 64 . . B B . . . −0.070.57 * * . −0.60 0.24 Gln 65 . . B B . . . 0.22 0.09 . * . 0.04 0.68 Val66 . . B B . . . 0.53 −0.21 * * . 0.98 0.68 Asn 67 . . . B T . . 1.42−0.60 * * F 2.32 2.12 Lys 68 . . . B T . . 1.42 −1.17 * . F 2.66 2.05Ser 69 . . . . T T . 1.23 −1.19 * . F 3.40 3.69 Asp 70 . . . . T T .0.34 −1.19 * . F 3.06 1.70 Asp 71 . . B . . T . 1.20 −0.90 * . F 2.170.60 Ser 72 . . B . . T . 0.39 −0.50 * . . 1.38 0.77 Val 73 . . B B . .. −0.47 −0.20 * . . 0.64 0.38 Ile 74 . . B B . . . −0.17 0.49 * . .−0.60 0.19 Gln 75 . . B B . . . −0.38 0.89 * . . −0.60 0.23 Leu 76 . . BB . . . −0.38 0.93 * . . −0.60 0.47 Leu 77 . . B B . . . 0.03 0.69 * . .−0.17 1.08 Asn 78 . . . . . T C 0.89 0.00 * . F 1.16 1.22 Pro 79 . . . .. T C 1.47 0.00 * . F 1.44 2.56 Asn 80 . . . . T T . 0.58 −0.20 * . F2.52 4.49 Arg 81 . . . . T T . 1.14 −0.20 * . F 2.80 1.96 Gln 82 . . B B. . . 1.26 0.16 . * F 1.12 1.98 Thr 83 . . B B . . . 1.37 0.51 . * F0.54 1.07 Ile 84 . . B B . . . 1.58 0.11 * * . 0.41 1.07 Tyr 85 . . B B. . . 0.88 0.11 * * . 0.13 1.03 Phe 86 . . B B . . . 0.88 0.50 . * .−0.60 0.62 Arg 87 . . B B . . . 0.67 0.01 * . . −0.15 1.73 Asp 88 . . BB . . . 0.17 −0.24 * . F 0.94 1.70 Phe 89 . . B . . . . 1.10 −0.31 * . F1.48 1.62 Arg 90 . . . . . . C 1.34 −1.10 * . F 2.32 1.66 Pro 91 . . . .. . C 1.74 −1.10 * * F 2.66 1.66 Leu 92 . . . . T T . 1.74 −0.71 * * F3.40 2.56 Lys 93 . . . . T T . 1.04 −1.50 . * F 3.06 2.56 Asp 94 . . . .T T . 1.74 −0.71 * * F 2.72 1.44 Ser 95 A . . . . T . 0.82 −0.74 * * F1.98 3.01 Arg 96 . A B . . . . 0.22 −0.74 . . F 1.24 1.24 Phe 97 . A B .. . . 1.03 −0.06 . . . 0.30 0.61 Gln 98 . A B . . . . 0.29 0.34 . . .−0.30 0.74 Leu 99 . A B . . . . −0.01 0.74 . * . −0.60 0.33 Leu 100 . AB . . . . −0.01 1.13 . * . −0.60 0.50 Asn 101 . A . . . . C −0.42 0.73. * . −0.40 0.39 Phe 102 . A . . . . C 0.28 0.71 * . F −0.25 0.63 Ser103 . . . . . T C −0.53 0.03 * . F 0.60 1.33 Ser 104 A . . . . T . 0.320.03 * * F 0.25 0.68 Ser 105 A . . . . T . 0.28 −0.37 . * F 1.00 1.58Glu 106 A . . . . T . −0.02 −0.51 . * F 1.15 0.87 Leu 107 A . . B . . .−0.13 −0.51 . * F 0.75 0.87 Lys 108 A . . B . . . −0.14 −0.21 . * F 0.450.54 Val 109 A . . B . . . 0.16 −0.11 * * . 0.30 0.45 Ser 110 . . B B .. . −0.40 0.29 * * . −0.30 0.87 Leu 111 . . B B . . . −0.70 0.24 . * .−0.30 0.32 Thr 112 . . B B . . . −0.78 0.63 . * . −0.60 0.58 Asn 113 . .B B . . . −1.12 0.67 . . . −0.60 0.31 Val 114 . . B B . . . −0.27 0.67 .. . −0.60 0.50 Ser 115 . . B B . . . 0.03 −0.01 . . . 0.64 0.58 Ile 116. . B B . . . 0.50 −0.50 . * F 1.13 0.62 Ser 117 . . B . . T . 0.92−0.47 * * F 1.87 0.83 Asp 118 . . . . T T . 0.68 −1.11 * * F 3.06 1.21Glu 119 . . . . T T . 0.83 −0.74 * * F 3.40 2.70 Gly 120 . . . . T T .0.47 −0.64 * * F 3.06 1.74 Arg 121 . . . B T . . 1.36 −0.46 * * F 1.870.56 Tyr 122 . . . B T . . 0.84 −0.06 * * . 1.38 0.56 Phe 123 . . B B .. . 0.60 0.63 * * . −0.26 0.47 Cys 124 . . B B . . . 0.29 0.96 * * .−0.60 0.37 Gln 125 . . B B . . . 0.63 1.44 * * . −0.60 0.34 Leu 126 . .B B . . . 0.31 0.69 * * . −0.60 0.66 Tyr 127 . . . . T . . 0.34 0.33 * .. 0.79 1.91 Thr 128 . . . . T . . 1.04 0.19 * . F 1.28 1.71 Asp 129 . .. . . T C 1.71 0.19 * . F 1.62 3.58 Pro 130 . . . . . T C 1.41 −0.50 * .F 2.86 3.96 Pro 131 . . . . T T . 1.98 −0.87 . . F 3.40 3.68 Gln 132 . .. . T T . 1.91 −0.60 . . F 3.06 3.45 Glu 133 . . B B . . . 1.91 −0.11 *. F 1.62 3.22 Ser 134 . . B B . . . 1.02 −0.06 * . F 1.28 3.01 Tyr 135 .. B B . . . 0.92 0.20 . . F 0.34 1.22 Thr 136 . . B B . . . 0.28 0.29 .. F 0.00 1.01 Thr 137 . . B B . . . −0.53 0.93 * . F −0.45 0.56 Ile 138. . B B . . . −1.39 1.23 . . . −0.60 0.30 Thr 139 . . B B . . . −1.301.11 . . . −0.60 0.15 Val 140 . . B B . . . −1.27 1.06 . * . −0.60 0.16Leu 141 . . B B . . . −0.84 1.00 . * . −0.60 0.36 Val 142 . . B B . . .−0.53 0.31 . * . −0.30 0.49 Pro 143 . . B . . T . −0.46 0.23 . * F 0.401.06 Pro 144 . . . . T T . −0.74 0.27 . . F 0.80 1.06 Arg 145 . . . . TT . −0.78 0.20 . . F 0.80 1.41 Asn 146 A . . . . T . 0.03 0.24 . . .0.10 0.64 Leu 147 . A B . . . . 0.00 −0.19 * * . 0.30 0.69 Met 148 . A B. . . . 0.21 0.07 * . . −0.30 0.25 Ile 149 . A B . . . . 0.47 0.47 * * .−0.60 0.27 Asp 150 . A B . . . . 0.36 0.07 * * . −0.30 0.64 Ile 151 A A. . . . . 0.04 −0.61 * * . 0.75 1.09 Gln 152 A . . . . T . 0.27 −0.74. * F 1.30 2.24 Lys 153 A . . . . T . 0.01 −0.93 . * F 1.30 1.36 Asp 154A . . . . T . 0.90 −0.29 . * F 1.00 1.43 Thr 155 A . . . . T . 0.56−0.97 . * F 1.30 1.43 Ala 156 A A . . . . . 1.44 −0.94 . . F 0.75 0.71Val 157 A A . . . . . 1.44 −0.94 . . F 0.75 0.74 Glu 158 A A . . . . .0.51 −0.94 . . F 0.75 0.88 Gly 159 A A . . . . . 0.51 −0.74 . * F 0.750.61 Glu 160 A A . . . . . −0.03 −1.24 . * F 0.90 1.43 Glu 161 A A . . .. . 0.56 −1.24 . * F 0.75 0.61 Ile 162 A A . . . . . 0.74 −0.84 . * F0.75 1.00 Glu 163 A A . . . . . 0.43 −0.70 . * . 0.60 0.31 Val 164 A A .. . . . 0.19 −0.21 . * . 0.30 0.26 Asn 165 A A . . . . . −0.41 0.29 . *. −0.30 0.37 Cys 166 A A . . . . . −1.00 0.21 . * . −0.30 0.21 Thr 167 AA . . . . . −0.41 0.71 . * . −0.60 0.29 Ala 168 A A . . . . . −0.37 0.46. * . −0.60 0.24 Met 169 A . . . . . . 0.28 0.06 . . . −0.10 0.90 Ala170 A . . . . . . −0.31 −0.09 * . . 0.50 0.96 Ser 171 A . . . . . . 0.04−0.07 . . F 0.65 0.96 Lys 172 A . . . . . . 0.04 −0.09 * . F 0.80 1.40Pro 173 A . . B . . . −0.26 −0.21 * * F 0.60 2.00 Ala 174 A . . B . . .0.46 −0.03 * * F 0.60 1.05 Thr 175 . . B B . . . 0.76 −0.41 * * F 0.601.02 Thr 176 . . B B . . . 0.36 0.50 * * F −0.45 0.70 Ile 177 . . B B .. . 0.36 0.86 * * . −0.60 0.60 Arg 178 . . B B . . . 0.22 0.36 * . .−0.30 0.83 Trp 179 . . B B . . . 0.81 0.30 * . . −0.30 0.57 Phe 180 . .. . . T C 0.81 0.21 * * . 0.45 1.30 Lys 181 . . . . . T C 1.12 0.01 * *F 0.45 0.96 Gly 182 . . . . . T C 1.20 0.01 * * F 0.60 1.58 Asn 183 . .. . . T C 1.13 −0.21 * * F 1.20 1.50 Thr 184 . A . . . . C 1.08 −1.00. * F 1.40 1.50 Glu 185 A A . . . . . 1.82 −0.57 . * F 1.50 1.50 Leu 186A A . . . . . 1.48 −1.00 . * F 1.80 1.87 Lys 187 A A . . . . . 1.82−1.01 . * F 2.10 1.74 Gly 188 . . . . . T C 0.97 −1.50 . * F 3.00 1.74Lys 189 . . . . . T C 1.28 −0.86 . * F 2.70 1.56 Ser 190 A . . . . T .1.28 −1.54 . * F 2.20 1.35 Glu 191 A . . . . T . 1.80 −1.54 . * F 1.902.37 Val 192 A . . . . . . 1.46 −1.06 * * F 1.40 1.25 Glu 193 A . . . .. . 1.80 −0.67 * * F 1.10 1.25 Glu 194 A . . . . . . 1.16 −1.06 * . F1.10 1.20 Trp 195 A . . . . T . 1.21 −0.44 * . F 1.00 1.60 Ser 196 A . .. . T . 0.90 −0.33 . . . 0.85 1.45 Asp 197 A . . . . T . 0.90 0.16 * . .0.25 1.21 Met 198 A . . . . T . 0.59 0.80 . . . −0.20 0.85 Tyr 199 A . .B . . . 0.29 0.37 . . . −0.30 0.92 Thr 200 A . . B . . . 0.58 0.37 * . .−0.30 0.74 Val 201 A . . B . . . 0.07 0.77 * . . −0.45 1.29 Thr 202 A .. B . . . −0.53 0.84 . . F −0.45 0.68 Ser 203 A A . B . . . −0.74 0.70. * F −0.45 0.47 Gln 204 A A . B . . . −0.46 0.90 * * F −0.45 0.52 Leu205 A A . B . . . −1.00 0.26 . * . −0.30 0.72 Met 206 A A . B . . .−0.18 0.41 * * . −0.60 0.40 Leu 207 A A . B . . . 0.18 0.53 * * . −0.600.31 Lys 208 A A . B . . . 0.48 0.13 * * . −0.30 0.76 Val 209 A A . . .. . 0.48 −0.56 * * . 1.09 1.32 His 210 A A . . . . . 1.29 −1.17 . * F1.58 2.68 Lys 211 A A . . . . . 1.54 −1.86 * . F 1.92 2.24 Glu 212 A . .. . T . 1.50 −1.43 . * F 2.66 2.98 Asp 213 . . . . T T . 1.24 −1.43 . .F 3.40 1.63 Asp 214 . . . . T T . 1.24 −1.50 * . F 3.06 1.26 Gly 215 . .. . T T . 0.39 −0.86 . . F 2.57 0.54 Val 216 . . B B . . . −0.32 −0.17 *. . 0.98 0.23 Pro 217 . . B B . . . −0.32 0.40 * . . −0.26 0.07 Val 218. . B B . . . −1.18 0.80 * . . −0.60 0.13 Ile 219 . . B B . . . −1.181.01 * . . −0.60 0.13 Cys 220 . . B B . . . −0.87 0.37 * . . −0.30 0.14Gln 221 . . B B . . . −0.22 0.44 * * . −0.60 0.26 Val 222 . . B B . . .−0.60 0.23 * . . −0.30 0.58 Glu 223 . . B B . . . −0.60 0.04 * . . −0.151.08 His 224 . . B . . . . −0.02 0.11 * . . −0.10 0.46 Pro 225 . . B . .. . 0.30 0.20 . . . −0.10 0.90 Ala 226 . . . . T . . 0.30 −0.01 . * .0.90 0.52 Val 227 . . . . T T C 0.34 0.39 . * . 0.50 0.61 Thr 228 . . .. . T C 0.34 0.57 . * F 0.15 0.33 Gly 229 . . . . . T C 0.07 0.54 . * F0.15 0.56 Asn 230 . . . . . T C 0.28 0.53 . * F 0.30 1.09 Leu 231 . . BB . . . 0.98 0.29 . . F 0.00 1.30 Gln 232 . . B B . . . 1.59 −0.20 . * F0.60 2.58 Thr 233 . . B B . . . 1.09 0.13 * . F 0.00 2.51 Gln 234 . . BB . . . 1.43 0.41 . . F −0.30 2.51 Arg 235 . . B B . . . 0.58 −0.27 . *F 0.60 2.51 Tyr 236 . . B B . . . 1.39 −0.03 . . . 0.45 1.29 Leu 237 . .B B . . . 1.14 −0.11 * * . 0.45 1.29 Glu 238 . . B B . . . 1.50 0.24 * *. −0.15 1.03 Val 239 . . B B . . . 1.29 0.24 * * . −0.15 1.32 Gln 240 .. . B T . . 1.18 −0.09 * * . 0.85 2.47 Tyr 241 . . . B T . . 0.57 −0.37. * . 0.85 2.47 Lys 242 . . . B . . C 1.34 0.27 . * F 0.20 2.47 Pro 243A . . B . . . 0.46 0.13 . * F 0.00 1.94 Gln 244 . . B B . . . 1.31 0.41. * . −0.60 0.87 Val 245 . . B B . . . 0.71 0.06 . * . −0.30 0.75 His246 . . B B . . . 0.64 0.67 . * . −0.60 0.48 Ile 247 . . B B . . . 0.360.73 . * . −0.60 0.40 Gln 248 . . B B . . . 0.36 1.09 . * . −0.60 0.85Met 249 . . B B . . . −0.46 0.87 . * . −0.60 0.96 Thr 250 . . B B . . .0.40 1.06 . * . −0.45 1.13 Tyr 251 . . B . . T . 0.09 0.77 . * . −0.051.13 Pro 252 . . B . . T . 0.17 0.80 . * . −0.05 1.13 Leu 253 . . . . TT . −0.14 0.87 * * . 0.20 0.65 Gln 254 . . B . . T . 0.57 0.87 * * .0.06 0.60 Gly 255 . . B . . . . 0.88 0.11 * . F 0.57 0.76 Leu 256 . . B. . . . 0.78 −0.31 * . F 1.58 1.59 Thr 257 . . B . . T . 0.99 −0.57 * .F 2.19 0.91 Arg 258 . . B . . T . 1.21 −0.97 * . F 2.60 1.53 Glu 259 A .. . . T . 0.40 −0.90 * . F 2.34 1.87 Gly 260 A . . . . T . 0.74 −0.90 *. F 2.08 1.07 Asp 261 A A . . . . . 0.74 −1.39 * . F 1.27 0.95 Ala 262 AA . . . . . 0.74 −0.70 * . F 1.01 0.45 Leu 263 A A . . . . . −0.03−0.21 * . . 0.30 0.66 Glu 264 A A . . . . . −0.03 −0.07 . * . 0.30 0.21Leu 265 A A . . . . . −0.28 −0.07 * . . 0.30 0.36 Thr 266 A A . . . . .−1.17 −0.07 * . . 0.30 0.44 Cys 267 A A . . . . . −0.92 −0.07 * * . 0.300.18 Glu 268 A A . . . . . −0.07 0.36 * * . −0.10 0.22 Ala 269 A A . . .. . −0.28 −0.33 * * . 0.70 0.30 Ile 270 A A . . . . . 0.53 −0.39 . * .0.90 0.86 Gly 271 . A . . T . . 0.63 −0.56 . . F 1.95 0.86 Lys 272 . . .. . . C 0.44 −0.13 * . F 2.00 1.32 Pro 273 . . . . . . C −0.16 0.01 * .F 1.20 1.39 Gln 274 . . . B . . C −0.42 −0.06 . . F 1.40 1.39 Pro 275 .. B B . . . 0.16 0.16 . . F 0.25 0.52 Val 276 . . B B . . . 0.21 0.64 .. . −0.40 0.48 Met 277 . . B B . . . −0.69 1.13 * . . −0.60 0.29 Val 278. . B B . . . −0.37 1.37 * * . −0.60 0.14 Thr 279 . . B B . . . −1.220.94 * * . −0.60 0.37 Trp 280 . . B B . . . −1.01 0.94 * * . −0.60 0.28Val 281 . . B B . . . −0.16 0.33 * * . −0.30 0.63 Arg 282 . . B B . . .0.44 −0.31 * * . 0.30 0.73 Val 283 A . . B . . . 0.70 −0.80 * * . 0.751.19 Asp 284 A . . B . . . 0.80 −1.10 * * F 0.90 1.59 Asp 285 . A . . T. . 1.09 −1.31 * * F 1.30 1.26 Glu 286 A A . . . . . 1.91 −0.91 * * F0.90 2.93 Met 287 A A . . . . . 1.21 −1.06 * * F 0.90 2.39 Pro 288 A A .. . . . 1.21 −0.56 . . F 0.90 1.45 Gln 289 A A . B . . . 0.40 0.09 . . .−0.30 0.62 His 290 A A . B . . . 0.10 0.77 . . . −0.60 0.52 Ala 291 . AB B . . . −0.24 0.54 . . . −0.60 0.45 Val 292 . A B B . . . 0.14 0.54 .. . −0.60 0.26 Leu 293 . A B B . . . 0.36 0.57 . . . −0.60 0.29 Ser 294. A . B . . C −0.46 0.47 * . F −0.25 0.46 Gly 295 . . . . . T C −1.120.66 * . F 0.15 0.51 Pro 296 . . . . . T C −1.42 0.80 * . F 0.15 0.54Asn 297 . . . . . T C −0.57 0.80 * . F 0.15 0.28 Leu 298 . . B . . T .0.24 0.81 * . . −0.20 0.46 Phe 299 . . B . . . . −0.27 0.79 * . . −0.400.48 Ile 300 . . B . . . . 0.08 1.04 * . . −0.40 0.24 Asn 301 . . B . .. . 0.33 1.04 * . . −0.40 0.48 Asn 302 . . B . . . . 0.02 0.36 * . .0.39 1.10 Leu 303 . . . . . . C 0.83 0.06 * . F 1.08 2.27 Asn 304 . . .. T . . 1.53 −0.63 * . F 2.52 2.36 Lys 305 . . . . T . . 2.08 −0.63 * .F 2.86 2.36 Thr 306 . . . . T T . 1.77 −0.60 * . F 3.40 2.83 Asp 307 . .. . T T . 1.52 −0.80 * * F 3.06 2.54 Asn 308 . . . . T T . 2.44−0.44 * * F 2.42 1.99 Gly 309 . . . . T T . 1.78 −0.44 * * F 2.08 2.70Thr 310 . . B . . . . 1.73 −0.36 . * F 0.99 0.87 Tyr 311 . . B . . . .1.46 −0.36 . * . 0.50 0.93 Arg 312 . . B . . . . 1.16 −0.26 . * . 0.500.95 Cys 313 . . B . . . . 1.16 −0.30 . * . 0.50 0.88 Glu 314 . . B . .. . 0.61 −0.39 . * . 0.50 0.91 Ala 315 A . . . . T . 0.07 −0.46 . * .0.70 0.32 Ser 316 A . . . . T . −0.03 0.19 . * . 0.10 0.45 Asn 317 A . .. . T . −0.10 0.04 . * . 0.10 0.26 Ile 318 A . . . . T . −0.02 0.04 * *. 0.10 0.51 Val 319 A . . . . . . −0.06 0.04 * . . 0.10 0.38 Gly 320 A .. . . . . 0.23 0.16 * * . 0.30 0.32 Lys 321 A . . . . . . 0.53 0.14 * *. 0.50 0.62 Ala 322 A . . . . . . 0.29 −0.54 * * F 1.90 1.40 His 323 . .B . . T . 0.58 −0.43 . * F 2.00 2.21 Ser 324 . . B . . T . 0.62 −0.24. * . 1.65 1.09 Asp 325 . . B . . T . 0.72 0.44 . * . 0.40 0.89 Tyr 326. . B . . T . −0.18 0.70 . * . 0.35 1.03 Met 327 . . B B . . . 0.17 0.84. . . −0.40 0.57 Leu 328 . . B B . . . 0.20 1.21 . . . −0.60 0.53 Tyr329 . . B B . . . 0.29 1.21 . . . −0.60 0.57 Val 330 . . B B . . . 0.080.89 . . . −0.60 0.89 Tyr 331 . . B . . . . 0.01 0.70 . . . −0.25 1.67Asp 332 . . B . . . . 0.30 0.50 * . F −0.10 1.54 Pro 333 . . B . . T .0.22 0.23 . . F 0.40 2.99 Pro 334 . . . . T T . 0.26 0.27 * . F 0.801.34 Thr 335 . . . . T T . 0.90 −0.06 * . F 1.40 1.24 Thr 336 . . B . .T . 0.93 0.37 . . F 0.40 1.24 Ile 337 . . B . . . . 0.62 0.37 . . F 0.321.24 Pro 338 . . B . . . . 0.52 0.43 . . F 0.14 1.24 Pro 339 . . . . . TC 0.42 0.43 . . F 0.66 1.24 Pro 340 . . . . . T C 0.42 0.43 . . F 0.782.55 Thr 341 . . . . . T C 0.42 0.23 . . F 1.20 2.38 Thr 342 . . B . . T. 1.00 0.29 . . F 0.88 2.22 Thr 343 . . B B . . . 0.90 0.34 . . F 0.362.07 Thr 344 . . B B . . . 0.80 0.40 . . F −0.06 2.07 Thr 345 . . B B .. . 0.70 0.40 . . F −0.18 2.07 Thr 346 . . B B . . . 0.70 0.40 . . F−0.30 2.07 Thr 347 . . B B . . . 0.70 0.40 . . F −0.30 2.07 Thr 348 . .B B . . . 0.70 0.40 . . F −0.30 2.07 Thr 349 . . B B . . . 0.70 0.40 . .F −0.30 2.07 Thr 350 . . B B . . . 0.12 0.40 . . F −0.30 2.07 Thr 351 .. B B . . . −0.38 0.60 . . F −0.30 1.01 Thr 352 . . B B . . . −0.38 0.80. . F −0.45 0.57 Thr 353 . . B B . . . −0.96 0.80 * . F −0.45 0.57 Ile354 . . B B . . . −1.53 1.00 * . . −0.60 0.28 Leu 355 . . B B . . .−1.53 1.20 * . . −0.60 0.14 Thr 356 . . B B . . . −1.22 1.20 * . . −0.600.14 Ile 357 . . B B . . . −1.21 0.71 * * . −0.60 0.32 Ile 358 . . B B .. . −0.79 0.41 . * . −0.26 0.53 Thr 359 . . B B . . . −0.49 −0.27 . * F1.13 0.71 Asp 360 . . B . . T . 0.43 −0.26 . * F 2.02 1.03 Ser 361 . . .. . T C 0.36 −0.94 . * F 2.86 2.87 Arg 362 . . . . T T . 0.86 −1.20 . *F 3.40 2.54 Ala 363 . . . . T T . 1.36 −1.26 . * . 2.91 1.95 Arg 364 . .. . T . . 1.28 −0.83 . * . 2.37 1.86

TABLE XI Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 A . .. . . . 0.14 0.06 . . . −0.10 0.89 Ser 2 A . . . . T . −0.28 0.01 . . .0.10 0.94 Ser 3 A . . . . T . 0.16 0.27 . . . 0.10 0.60 Ser 4 A . . . .T . 0.51 −0.16 * . . 0.85 1.22 Ser 5 A . . . . T . 0.09 −0.27 * . F 1.001.24 Leu 6 A A . . . . . −0.12 0.03 * . F −0.15 0.76 Lys 7 A A . . . . .−0.49 0.33 * . F −0.15 0.47 His 8 A A . . . . . −0.79 0.51 * . . −0.600.19 Leu 9 A A . . . . . −1.08 0.74 * . . −0.60 0.23 Leu 10 A A . . . .. −1.59 0.56 * * . −0.60 0.11 Cys 11 A A . . . . . −1.08 1.24 * * .−0.60 0.07 Met 12 A A . . . . . −1.41 1.13 * . . −0.60 0.11 Ala 13 A A .. . . . −2.08 1.36 * . . −0.60 0.14 Leu 14 A A . . . . . −1.57 1.46 * .. −0.60 0.23 Ser 15 A . . B . . . −1.06 1.27 * . . −0.60 0.31 Trp 16 A .. B . . . −1.09 1.04 * . . −0.60 0.41 Phe 17 A . . B . . . −1.38 1.33 *. . −0.60 0.43 Ser 18 . . . B . . C −1.09 1.33 * . . −0.40 0.23 Ser 19 .. . B . . C −0.62 1.33 * . . −0.40 0.29 Phe 20 . . . B . . C −0.32 0.84. . . −0.40 0.33 Ile 21 . . . B . . C −0.34 0.06 . . . −0.10 0.43 Ser 22. . . B . . C 0.06 0.16 * * F 0.05 0.46 Gly 23 . . . . . . C −0.34 0.16. * F 0.25 0.71 Glu 24 . . . . . . C −0.34 0.16 . * F 0.25 0.88 Thr 25 .. . . . . C −0.46 −0.14 . * F 0.85 0.88 Ser 26 . . . . . . C −0.38 0.16. * F 0.25 0.73 Phe 27 A . . . . . . −0.08 0.41 * * . −0.40 0.35 Ser 28A . . . . . . −0.03 0.81 * * . −0.40 0.39 Leu 29 . . . . . . C −0.730.71 * . . −0.20 0.39 Leu 30 . . . . . . C −1.12 1.11 . . . −0.20 0.39Asn 31 . . . . T T . −1.63 1.11 . . . 0.20 0.25 Ser 32 . . . . T T .−1.14 1.41 * . . 0.20 0.25 Phe 33 . . . . T T . −1.09 1.16 . . . 0.200.47 Phe 34 . . B . . T . −0.49 1.23 . . . −0.20 0.46 Leu 35 . . . . . .C 0.02 1.26 . . . −0.20 0.53 Pro 36 . . . . T . . −0.28 1.26 . . . 0.000.82 Tyr 37 . . . . T T . 0.13 0.86 . * F 0.67 1.27 Pro 38 . . . . T T .0.17 0.07 . . F 1.14 3.02 Ser 39 . . . . T T . 0.20 −0.04 . * F 1.911.05 Ser 40 . . . . T T . 0.34 0.10 . . F 1.33 0.36 Arg 41 . . . B T . .−0.14 −0.09 . . F 1.70 0.12 Cys 42 . . . B T . . −0.20 0.27 . . . 0.780.08 Cys 43 . . . B T . . −0.84 0.27 . * . 0.61 0.08 Cys 44 . . . B T .. −0.54 0.53 . * . 0.14 0.03 Phe 45 . . . B T . . −0.91 0.93 . * . −0.030.10 Ser 46 . . . B T . . −1.32 0.93 . * . −0.20 0.10 Val 47 . . . B T .. −1.54 0.74 . * . −0.20 0.25 Gln 48 . . . B T . . −1.69 0.86 . * .−0.20 0.20 Cys 49 . . . B T . . −1.02 0.76 . * . −0.20 0.12 Ser 50 . . .B T . . −0.53 0.37 * * . 0.10 0.28 Ile 51 . . . B T . . −0.93 0.16 * * .0.10 0.25 Leu 52 . . . B T . . −0.38 0.54 * * . −0.20 0.40 Asp 53 . . .. . T C −1.04 0.36 . . . 0.30 0.40 Pro 54 . . . . T T . −0.38 0.54 * . F0.35 0.30 Phe 55 . . . . T T . −0.38 0.26 . . . 0.50 0.59 Ser 56 . . . .T T . −0.09 −0.04 * . . 1.10 0.48 Cys 57 . . . . T T . 0.83 0.57 * * .0.20 0.30 Asn 58 . . . . T T . 0.13 0.14 * * . 0.50 0.69 Ser 59 . . . .T T . 0.13 0.14 * * . 0.50 0.44 Met 60 . . . . T T . 0.54 0.19 * * .0.86 1.28 Arg 61 . . . . . . C 0.84 0.53 * * . 0.22 0.84 Phe 62 . . . .. . C 1.51 0.13 * * . 0.88 1.08 Pro 63 . . . . T . . 1.12 0.14 * * .1.29 1.76 Trp 64 . . . . T . . 1.03 −0.04 . * . 2.10 1.15 Glu 65 A . . .. . . 1.24 0.39 . * . 0.89 1.70 Asn 66 . . . . T . . 0.74 0.03 . * .1.08 1.40

TABLE XII Res I II III IV V VI VII VIII IX X XI XII XIII XIV Met 1 . . B. . . . 0.97 −0.71 * . . 1.64 1.91 Ser 2 . . B . . T . 0.76 −0.76 . . .2.07 2.00 Arg 3 . . B . . T . 0.33 −0.57 . . . 2.30 1.55 Arg 4 . . B . .T . −0.09 −0.31 . . . 1.77 1.29 Ser 5 . . B . . T . −0.29 −0.24 . . .1.39 0.79 Met 6 . A B . . . . 0.02 −0.13 . . . 0.76 0.41 Leu 7 . A B . .. . −0.27 0.79 . . . −0.37 0.22 Leu 8 . A B . . . . −1.19 1.29 . . .−0.60 0.17 Ala 9 . A B . . . . −1.51 1.59 . . . −0.60 0.14 Trp 10 . A B. . . . −1.51 1.40 . . . −0.60 0.26 Ala 11 . A B . . . . −1.72 1.10 . .. −0.60 0.42 Leu 12 . . B . . T . −1.72 1.10 * * . −0.20 0.34 Pro 13 . .B . . T . −0.80 1.29 * * . −0.20 0.27 Ser 14 . . B . . T . −1.020.37 * * . 0.10 0.52 Leu 15 . . B . . T . −1.08 0.56 * * . −0.20 0.52Leu 16 . A B . . . . −1.08 0.30 * * . −0.30 0.33 Arg 17 . A B . . . .−0.86 0.37 * * . −0.30 0.25 Leu 18 . A B . . . . −0.64 0.49 . * . −0.600.31 Gly 19 . A . . . . C −0.34 0.20 . * . −0.10 0.65 Ala 20 . A . . . .C 0.16 −0.49 . * . 0.50 0.57 Ala 21 . A . . . . C 0.97 0.00 . * . 0.651.00 Gln 22 . A B . . . . 0.86 −0.69 . * F 1.21 1.75 Glu 23 . A B . . .. 1.46 −1.11 . . F 1.52 2.90 Thr 24 . A . . T . . 1.21 −1.19 . . F 2.234.44 Glu 25 . A . . T . . 1.13 −1.19 . . F 2.54 2.59 Asp 26 . . . . T T. 1.06 −1.01 . . F 3.10 0.80 Pro 27 . . . . T T . 0.76 −0.44 . . F 2.490.30 Ala 28 . . . . T T . 0.54 −0.54 . . . 2.33 0.23 Cys 29 . . . . T T. −0.03 −0.11 . . . 1.72 0.21 Cys 30 . . B B . . . −0.89 0.57 . . .−0.29 0.10 Ser 31 . . B B . . . −1.10 0.79 . . . −0.60 0.07 Pro 32 . . BB . . . −0.78 0.71 . . . −0.60 0.20 Ile 33 . . B B . . . −0.19 0.14 . .. 0.00 0.75 Val 34 . . B . . T . 0.48 −0.03 . * F 1.45 0.90 Pro 35 . . B. . T . 0.86 −0.41 . * F 1.90 1.01 Arg 36 . . . . T T . 1.20 0.07 . * F2.00 1.51 Asn 37 . . . . . T C 0.82 −0.61 * * F 3.00 4.07 Glu 38 . A . .T . . 0.90 −0.76 * * F 2.50 2.66 Trp 39 . A . . T . . 1.17 −0.50 * * F2.20 1.12 Lys 40 . A . . . . C 1.08 0.00 * * . 1.10 0.70 Ala 41 . A . .. . C 0.97 −0.01 * * . 0.80 0.54 Leu 42 . A . . . . C 0.30 −0.01 * . .0.50 0.90 Ala 43 A A . . . . . −0.29 −0.36 * * . 0.30 0.24 Ser 44 A A .. . . . 0.00 0.14 * . . −0.30 0.24 Glu 45 A A . . . . . −0.08 0.04 * . .−0.30 0.50 Cys 46 A A . . . . . −0.30 −0.14 * . . 0.30 0.68 Ala 47 A A .. . . . 0.21 0.04 . . . −0.30 0.42 Gln 48 . A B . . . . −0.01 0.04 . . .−0.30 0.32 His 49 . A B . . . . 0.08 0.73 * * . −0.60 0.50 Leu 50 . A B. . . . −0.73 0.59 * * . −0.60 0.76 Ser 51 . A B . . . . 0.04 0.77 * * .−0.60 0.36 Leu 52 . . B . . . . 0.39 0.37 * * . −0.10 0.52 Pro 53 . . BB . . . −0.47 0.63 * * . −0.60 0.99 Leu 54 . . B B . . . −1.29 0.59 . *. −0.60 0.55 Arg 55 . . B B . . . −1.33 0.84 . * . −0.60 0.49 Tyr 56 . .B B . . . −1.33 0.80 . * . −0.60 0.24 Val 57 . . B B . . . −0.56 0.76. * . −0.60 0.39 Val 58 . . B B . . . −0.66 0.57 . * . −0.60 0.27 Val 59. . B B . . . −0.43 1.06 . * . −0.60 0.25 Ser 60 . . B . . . . −0.890.80 . . . −0.40 0.34 His 61 . . B . . . . −0.94 0.59 . . . −0.40 0.45Thr 62 . . B . . . . −0.39 0.33 . . . −0.10 0.81 Ala 63 . . . . T . .−0.20 0.07 . . F 0.45 0.81 Gly 64 . . . . T T . 0.66 0.26 . . F 0.650.32 Ser 65 . . . . T T . 0.64 0.16 . . F 0.65 0.35 Ser 66 . . . . T T .0.47 0.16 . . F 0.65 0.51 Cys 67 . . . . T T . 0.19 0.09 . . F 0.65 0.79Asn 68 . . . . T . . 0.48 0.16 . . F 0.45 0.60 Thr 69 . . . . . . C 0.160.16 . . F 0.25 0.60 Pro 70 . . . . T T . 0.46 0.34 . . F 0.65 0.60 Ala71 . . . . T T . 0.76 0.17 . * F 0.65 0.64 Ser 72 . . B . . T . 1.420.17 . * F 0.25 0.77 Cys 73 . . B . . T . 0.83 0.09 * * F 0.25 0.86 Gln74 . A B . . . . 1.26 0.16 . * F −0.15 0.86 Gln 75 . A B . . . . 1.47−0.34 * * F 0.60 1.26 Gln 76 . A B . . . . 1.20 −0.33 * * F 0.60 3.79Ala 77 . A B . . . . 1.50 −0.26 * . F 0.60 1.62 Arg 78 . A B . . . .2.13 −0.26 * . F 0.60 1.62 Asn 79 . A B . . . . 1.89 −0.16 * . . 0.451.28 Val 80 . A B . . . . 1.86 0.20 * . . −0.15 1.98 Gln 81 . A B . . .. 1.26 0.20 * * . −0.15 1.37 His 82 . A B . . . . 1.89 0.81 * * . −0.600.85 Tyr 83 . . B . . . . 1.47 0.41 * . . −0.25 2.28 His 84 . . B . . .. 0.66 0.26 . . . 0.05 1.90 Met 85 . . B B . . . 1.17 0.54 . . . −0.451.15 Lys 86 . . B B . . . 0.88 0.47 . . . −0.60 0.73 Thr 87 . . . B T .. 0.24 0.63 . . . −0.20 0.56 Leu 88 . . . B T . . 0.49 0.70 . * . −0.200.30 Gly 89 . . . B T . . −0.33 0.09 . . . 0.10 0.25 Trp 90 . . B B . .. −0.08 0.73 . . . −0.60 0.13 Cys 91 . . B B . . . −0.37 0.67 . . .−0.60 0.16 Asp 92 . . B . . T . −0.06 0.74 . * . −0.20 0.25 Val 93 . . B. . T . 0.06 0.71 * . . −0.20 0.38 Gly 94 . . B . . T . −0.41 0.59 . * .−0.20 0.61 Tyr 95 . . B . . T . −1.01 0.70 . * . −0.20 0.30 Asn 96 . . BB . . . −0.69 1.39 . * . −0.60 0.29 Phe 97 . . B B . . . −0.69 1.17 . *. −0.60 0.29 Leu 98 . . B B . . . 0.17 0.74 . * . −0.60 0.32 Ile 99 . .B B . . . 0.17 −0.01 . . . 0.30 0.33 Gly 100 . . B . . T . −0.40 0.01 .. . 0.10 0.38 Glu 101 . . B . . T . −1.26 −0.09 . . F 0.85 0.38 Asp 102. . . . T T . −0.80 −0.13 . . F 1.25 0.40 Gly 103 . . . . . T C 0.01−0.06 . . F 1.05 0.63 Leu 104 . . B . . . . 0.56 −0.49 * * . 0.50 0.63Val 105 . . B . . . . 1.01 −0.06 * * . 0.78 0.37 Tyr 106 . . B . . . .0.67 −0.06 * * . 1.06 0.74 Glu 107 . . B . . . . 0.38 −0.06 * . F 1.490.89 Gly 108 . . . . T T . 0.72 0.17 . . F 1.92 1.25 Arg 109 . . . . T T. 0.83 −0.07 . * F 2.80 1.29 Gly 110 . . . . T T . 1.38 −0.04 . . F 2.370.64 Trp 111 . . . . T T . 1.28 0.44 . * . 1.04 0.94 Asn 112 . . . . . .C 0.69 0.44 . . . 0.36 0.47 Phe 113 . . B . . . . 1.00 0.94 . . . −0.120.48 Thr 114 . . . . . . C 0.59 1.01 . . . −0.20 0.63 Gly 115 . . . . .. C 0.59 0.49 . * . −0.20 0.52 Ala 116 . . . . . . C 0.84 0.51 . * .−0.20 0.60 His 117 . . . . . T C 0.03 0.23 . . . 0.30 0.56 Ser 118 . . .. . T C 0.44 0.43 . . . 0.00 0.47 Gly 119 . . . . . T C 0.76 0.91 . . .0.00 0.49 His 120 . . . . . T C 0.89 0.81 . . . 0.00 0.58 Leu 121 . . .. T . . 0.88 0.74 . . . 0.00 0.67 Trp 122 . . . . . . C 0.61 0.97 . . .−0.20 0.67 Asn 123 . . . . . . C 0.02 0.93 . . . −0.20 0.66 Pro 124 . .B B . . . 0.02 1.11 . * . −0.60 0.56 Met 125 . . . B T . . −0.830.86 * * . −0.20 0.52 Ser 126 . . B B . . . −0.32 0.63 . * . −0.60 0.23Ile 127 . . B B . . . −0.73 0.61 . * . −0.60 0.20 Gly 128 . . B B . . .−1.33 0.97 . * . −0.60 0.17 Ile 129 . . B B . . . −1.47 0.97 . * . −0.600.13 Ser 130 . . B B . . . −0.87 1.01 . * . −0.60 0.18 Phe 131 . . B B .. . −0.81 0.73 . * . −0.60 0.29 Met 132 . . B . . T . −0.52 1.06 . * .−0.20 0.66 Gly 133 . . . . T T . −0.18 0.99 * * . 0.20 0.48 Asn 134 . .. . T T . 0.82 0.60 * * . 0.20 0.93 Tyr 135 . . . . T T . 0.27 −0.19 * *. 1.25 1.85 Met 136 . . . . T . . 0.76 −0.16 * * . 1.31 1.39 Asp 137 . .. . T . . 1.04 −0.16 * * . 1.57 1.33 Arg 138 . . B . . . . 1.18−0.07 * * F 1.58 1.23 Val 139 . . B . . T . 1.18 −0.40 * . F 2.04 1.92Pro 140 . . B . . T . 0.83 −0.61 . . F 2.60 1.99 Thr 141 . . . . . T C0.54 −0.11 . * F 2.24 1.03 Pro 142 . . B . . T . 0.66 0.57 . * F 0.730.97 Gln 143 . A B . . . . −0.04 −0.07 * * F 1.12 1.23 Ala 144 . A B . .. . 0.22 0.00 * . . 0.56 0.86 Ile 145 . A B . . . . 0.43 0.01 * . .−0.30 0.56 Arg 146 . A B . . . . 0.40 −0.01 * . . 0.30 0.56 Ala 147 . AB . . . . −0.20 0.01 * . . −0.30 0.55 Ala 148 . A B . . . . −1.01 0.20 *. . −0.30 0.65 Gln 149 . A B . . . . −1.01 0.20 * * . −0.30 0.27 Gly 150. A B . . . . −0.79 0.70 * * . −0.60 0.27 Leu 151 . A B . . . . −1.240.77 * . . −0.60 0.14 Leu 152 . A B . . . . −1.51 0.70 . . . −0.60 0.08Ala 153 . A B . . . . −1.51 0.94 . . . −0.60 0.06 Cys 154 . A B . . . .−1.51 1.01 . . . −0.60 0.08 Gly 155 . A B . . . . −1.51 0.73 . . . −0.600.16 Val 156 . A B . . . . −1.29 0.47 . . . −0.60 0.16 Ala 157 . A B . .. . −1.29 0.47 * * . −0.60 0.29 Gln 158 . A B . . . . −0.59 0.59 * * .−0.60 0.25 Gly 159 . A B . . . . −0.22 0.16 * * . −0.30 0.65 Ala 160 . AB . . . . 0.12 −0.10 * * F 0.45 0.86 Leu 161 . A B . . . . 0.73−0.20 * * F 0.45 0.80 Arg 162 . . B . . T . 0.47 0.16 * * F 0.40 1.26Ser 163 . . B . . T . −0.34 0.37 * * F 0.25 0.93 Asn 164 . . B . . T .0.04 0.56 . * F −0.05 0.93 Tyr 165 . . B . . T . 0.29 −0.13 . * . 0.700.95 Val 166 . . B B . . . 1.07 0.30 . * . −0.12 0.70 Leu 167 . . B B .. . 1.07 0.41 * * . −0.24 0.59 Lys 168 . . B B . . . 1.37 0.01 * . F0.39 0.74 Gly 169 . . B . . . . 0.51 −0.74 * * F 1.82 1.67 His 170 . . BB . . . 0.76 −0.74 * . F 1.80 1.50 Arg 171 . . B B . . . 1.72 −1.03 * .F 1.62 1.30 Asp 172 . . B B . . . 2.22 −1.03 * . F 1.44 2.57 Val 173 . .B B . . . 1.37 −0.97 * . F 1.26 2.72 Gln 174 . . B B . . . 1.41 −0.79 *. F 1.08 1.15 Arg 175 . . B B . . . 1.23 −0.40 * . F 0.57 0.92 Thr 176 .. B B . . . 0.78 0.03 * . F 0.24 1.92 Leu 177 . . . B . . C 0.78 −0.19 *. F 1.16 1.10 Ser 178 . . . . . T C 1.63 −0.19 * . F 1.53 0.90 Pro 179 .. . . . T C 0.82 0.21 * . F 1.20 1.08 Gly 180 . . . . T T . 0.47 0.41 *. F 0.98 1.08 Asn 181 . . . . T T . 0.74 0.49 . . F 0.86 1.26 Gln 182 .A B . . . . 0.74 0.60 * . F −0.06 1.11 Leu 183 . A B . . . . 0.16 0.86 *. . −0.48 0.93 Tyr 184 . A B . . . . 0.37 1.11 * . . −0.60 0.40 His 185. A B . . . . 0.71 1.11 * . . −0.60 0.40 Leu 186 . A B . . . . 0.421.11 * . . −0.60 0.79 Ile 187 . A B . . . . 0.21 1.34 * . . −0.60 0.53Gln 188 . A B . . . . 0.99 1.01 * . . −0.60 0.60 Asn 189 . A . . T . .0.99 1.01 . * . −0.20 0.99 Trp 190 . . . . . T C 1.13 1.09 . * . 0.152.21 Pro 191 . . . . . T C 1.64 0.40 . * . 0.45 2.50 His 192 . . . . T T. 2.32 0.39 . * . 0.86 2.09 Tyr 193 . . . . T T . 1.93 0.41 . . . 0.773.07 Arg 194 . . . . T . . 1.54 −0.07 . . . 1.68 2.54 Ser 195 . . . . .. C 1.44 −0.07 . . . 1.69 2.38 Pro 196 . . . . T . . 1.27 −0.14 . * .2.10 1.94

TABLE XIII Met 1 . A B . . . . −1.30 0.70 . . . −0.60 0.39 Leu 2 . A B .. . . −1.72 0.96 . . . −0.60 0.25 Leu 3 . A B . . . . −2.14 1.21 . . .−0.60 0.16 Pro 4 . A B . . . . −2.06 1.47 . . . −0.60 0.14 Leu 5 . A B .. . . −1.97 1.24 * . . −0.60 0.22 Leu 6 . A B . . . . −2.18 0.94 . . .−0.60 0.36 Leu 7 . A B . . . . −2.18 0.94 . . . −0.60 0.19 Ser 8 . A B .. . . −1.71 1.20 * . . −0.60 0.19 Ser 9 . . B B . . . −1.84 0.94 . . F−0.45 0.23 Leu 10 . . B B . . . −1.33 0.69 . . F −0.45 0.27 Leu 11 . . .B . . C −0.52 0.39 * . F 0.05 0.27 Gly 12 . . . . . T C −0.30 0.40 . . F0.45 0.35 Gly 13 . . . . . T C −0.60 0.51 . . F 0.15 0.43 Ser 14 . . B .. T . −0.30 0.44 . . F 0.12 0.52 Gln 15 . . B . . T . 0.17 −0.24 . * F1.19 0.88 Ala 16 . . B . . . . 1.09 −0.24 . * F 1.16 0.88 Met 17 . . B .. T . 0.73 −0.67 . * F 1.98 1.28 Asp 18 . . B . . T . 0.79 −0.27 . * F1.70 0.64 Gly 19 . . . . T T . 0.20 0.24 * * F 1.33 0.67 Arg 20 . . . .T T . 0.31 0.43 * * . 0.71 0.47 Phe 21 . . B B . . . 0.04 −0.19 * * .0.64 0.56 Trp 22 . . B B . . . 0.64 0.46 * * . −0.43 0.42 Ile 23 . . B B. . . 0.64 0.43 * * . −0.60 0.37 Arg 24 . . B B . . . 0.69 0.43 * * .−0.60 0.74 Val 25 . . B B . . . −0.28 0.03 * * . −0.30 0.94 Gln 26 . . BB . . . −0.18 −0.24 * * F 0.45 0.99 Glu 27 . . . B . . C −0.74 −0.31 * *F 0.65 0.50 Ser 28 . . . B . . C −0.07 0.33 . * . −0.10 0.50 Val 29 . .B B . . . −0.18 0.11 . * . −0.30 0.45 Met 30 . . B B . . . 0.33 −0.29 *. . 0.30 0.45 Val 31 . . B . . T . −0.48 0.14 . . . 0.10 0.33 Pro 32 . .B . . T . −1.14 0.44 . . . −0.20 0.37 Glu 33 . . . . T T . −1.73 0.37 .. F 0.65 0.20 Gly 34 . . . . T T . −1.18 0.44 . . F 0.35 0.19 Leu 35 . .B B . . . −1.43 0.19 . . . −0.30 0.16 Cys 36 . . B B . . . −0.79 0.40 .. . −0.30 0.07 Ile 37 . . B B . . . −1.24 0.83 . . . −0.60 0.11 Ser 38 .. B B . . . −1.54 0.97 . * . −0.60 0.07 Val 39 . . B . . T . −1.90 0.67. * . −0.20 0.18 Pro 40 . . B . . T . −1.39 0.89 . * . −0.20 0.22 Cys 41. . . . T T . −0.97 0.59 . * . 0.20 0.22 Ser 42 . . . . T T . −0.29 0.96. * . 0.20 0.46 Phe 43 . . . . T . . 0.12 0.74 * . . 0.28 0.46 Ser 44 .. B . . . . 0.98 0.31 . . . 0.61 1.69 Tyr 45 . . B . . T . 1.19 0.14 . .. 1.09 2.19 Pro 46 . . . . T T . 1.57 −0.24 . . F 2.52 4.22 Arg 47 . . .. T T . 1.56 −0.11 . . F 2.80 3.31 Gln 48 . . . . T T . 1.91 −0.01 . * F2.52 3.05 Asp 49 . . . . T . . 1.91 −0.34 . * F 2.04 1.95 Trp 50 . . . .T T . 1.84 −0.39 . * F 1.96 1.33 Thr 51 . . . . . T C 1.84 0.10 . * F0.88 1.11 Gly 52 . . . . T T . 1.14 0.13 . * F 0.80 1.03 Ser 53 . . . .. T C 0.90 0.63 . . F 0.15 0.99 Thr 54 . . . . . . C 0.56 0.47 . . F0.10 1.07 Pro 55 . . . . . T C 0.60 0.41 . . F 0.30 1.07 Ala 56 . . . .T T . 0.62 0.74 . . . 0.35 1.26 Tyr 57 . . . . T T . 0.27 1.27 . . .0.20 0.91 Gly 58 . . . . T T . 0.61 1.57 . . . 0.20 0.51 Tyr 59 . . B B. . . 0.33 1.14 * . . −0.45 1.01 Trp 60 . . B B . . . −0.31 1.14 * . .−0.60 0.65 Phe 61 . . B B . . . −0.03 1.03 * . . −0.60 0.49 Lys 62 . . BB . . . 0.21 1.09 * . . −0.60 0.45 Ala 63 . . B B . . . 0.24 0.33 * . .−0.30 0.74 Val 64 . . B B . . . 0.18 −0.10 * . . 0.79 1.24 Thr 65 . . BB . . . 0.51 −0.40 * . F 1.13 0.89 Glu 66 . . B B . . . 0.87 −0.40 * . F1.62 1.77 Thr 67 . . . B T . . 0.23 −0.47 * . F 2.36 2.36 Thr 68 . . . .T T . 0.61 −0.61 * . F 3.40 1.65 Lys 69 . . . . T T . 0.61 −0.67 * . F3.06 1.48 Gly 70 . . . . . T C 0.33 −0.03 . . F 2.07 0.76 Ala 71 . . . .. T C 0.02 −0.01 * . F 1.73 0.53 Pro 72 . . B . . . . 0.33 −0.01 * . .0.84 0.38 Val 73 . . B . . . . 0.61 0.39 . . . −0.10 0.62 Ala 74 . . B .. . . 0.57 0.46 . . . −0.10 0.84 Thr 75 . . B . . . . 0.61 0.36 * * .0.50 0.94 Asn 76 . . . . . . C 1.31 0.31 . . F 1.30 1.70 His 77 . . . .. T C 1.52 −0.33 * . F 2.40 3.29 Gln 78 . . . . . T C 1.52 −0.83 * . F3.00 3.95 Ser 79 . . . . . T C 2.11 −0.67 * . F 2.70 1.82 Arg 80 . . B .. T . 1.82 −1.07 * . F 2.20 2.32 Glu 81 . A B . . . . 1.52 −0.96 * . F1.50 1.33 Val 82 . A B . . . . 1.24 −0.97 * * F 1.54 1.33 Glu 83 . A B .. . . 1.36 −0.87 * * . 1.28 0.98 Met 84 . A B . . . . 1.31 −0.87 * * .1.77 1.10 Ser 85 . . . . . T C 1.31 −0.44 * * F 2.56 1.47 Thr 86 . . . .T T . 0.61 −1.09 . * F 3.40 1.67 Arg 87 . . . . T T . 1.47 −0.30 . * F2.76 1.46 Gly 88 . . . . T T . 0.66 −0.51 . * F 2.72 1.88 Arg 89 . . B B. . . 0.94 −0.21 . * F 1.28 1.08 Phe 90 . . B B . . . 0.90 −0.21 . * .0.64 0.79 Gln 91 . . B B . . . 1.21 0.21 . * . −0.30 0.79 Leu 92 . . B B. . . 0.89 −0.21 . * . 0.30 0.68 Thr 93 . . B B . . . 0.64 0.21 . * F0.34 1.21 Gly 94 . . . B . . C 0.58 −0.07 . * F 1.33 0.70 Asp 95 . . . .. T C 0.93 −0.47 * * F 2.22 1.71 Pro 96 . . . . . T C 0.93 −0.73 . * F2.86 1.17 Ala 97 . . . . T T . 1.08 −0.81 . . F 3.40 1.90 Lys 98 . . . .T T . 1.09 −0.67 . * F 2.91 0.61 Gly 99 . . . . T T . 0.62 −0.29 * . F2.27 0.53 Asn 100 . . B . T T . −0.23 −0.03 . * F 1.93 0.43 Cys 101 . .B . . T . −0.91 0.11 * * . 0.44 0.16 Ser 102 . . B . . T . −0.210.80 * * . −0.20 0.11 Leu 103 . A B B . . . −0.26 0.37 * * . −0.30 0.14Val 104 . A B B . . . −0.50 −0.03 * * . 0.30 0.43 Ile 105 . A B B . . .−0.50 −0.10 * * . 0.30 0.33 Arg 106 . A B B . . . −0.43 −0.09 * . . 0.300.68 Asp 107 . A B B . . . −0.13 −0.16 * . . 0.30 0.91 Ala 108 . A B . .. . 0.68 −0.40 * . . 0.45 2.25 Gln 109 . A B . . . . 1.53 −1.09 * . .1.03 1.92 Met 110 . A . . . . C 2.12 −1.09 . . . 1.51 1.99 Gln 111 . A B. . . . 2.01 −0.70 . . F 1.74 2.64 Asp 112 . . . . T T . 1.77 −0.80 . *F 2.82 2.64 Glu 113 . . . . T T . 1.66 −0.44 * . F 2.80 4.18 Ser 114 . .. . T T . 0.96 −0.27 * * F 2.52 2.09 Gln 115 . . . . T T . 1.67 0.11 * *F 1.64 1.08 Tyr 116 . . B B . . . 0.81 0.11 * * . 0.41 1.22 Phe 117 . .B B . . . 0.81 0.76 * . . −0.32 0.68 Phe 118 . . B B . . . 0.92 0.37 * *. 0.04 0.68 Arg 119 . . B B . . . 0.88 −0.03 * . . 0.98 0.85 Val 120 . .B B . . . 0.58 −0.36 * . . 1.32 0.97 Glu 121 . . . . T T . 0.58 −0.76 *. F 3.06 1.50 Arg 122 . . . . T T . 0.42 −0.79 * * F 3.40 1.20 Gly 123 .. . . T T . 1.23 −0.14 * * F 2.76 1.20 Ser 124 . . . . T T . 0.88−0.79 * * F 2.72 1.36 Tyr 125 . . B . . . . 1.73 −0.03 . * . 1.33 1.09Val 126 . . B . . . . 1.03 0.37 . * . 0.39 1.76 Arg 127 . . B . . . .0.32 0.73 . . . −0.25 1.14 Tyr 128 . . B . . . . 0.67 0.96 . * . −0.400.72 Asn 129 . . B . . . . 0.97 0.60 . * . −0.25 1.56 Phe 130 . . B . .. . 0.87 −0.04 . * . 0.65 1.33 Met 131 . . B . . . . 1.02 0.39 . * .−0.10 0.84 Asn 132 . . . . T T . 0.21 0.41 . * . 0.20 0.45 Asp 133 . . .. T T . −0.36 0.80 . . . 0.20 0.45 Gly 134 . . . . T T . −0.31 0.70 . *. 0.20 0.38 Phe 135 . . B . . T . −0.47 0.09 . . . 0.10 0.47 Phe 136 . .B B . . . −0.18 0.33 . * . −0.30 0.21 Leu 137 . . B B . . . −0.77 0.81 .. . −0.60 0.30 Lys 138 . . B B . . . −1.58 0.89 . . . −0.60 0.35 Val 139. . B B . . . −1.54 0.79 . * . −0.60 0.34 Thr 140 . . B B . . . −0.840.49 * * . −0.60 0.59 Ala 141 . . B B . . . −0.10 0.20 * . . −0.30 0.51Leu 142 . . B B . . . 0.50 0.20 . * F 0.00 1.38 Thr 143 . . B B . . .0.46 −0.01 . * F 0.85 1.48 Gln 144 . . B B . . . 0.46 −0.50 . . F 1.402.44 Lys 145 . . B . . T . 0.52 −0.36 . * F 1.75 2.20 Pro 146 . . B . .T . 0.22 −0.29 . * F 2.00 2.38 Asp 147 . . . . T T . 0.82 −0.09 * * F2.50 0.97 Val 148 . . B . . T . 1.13 −0.06 . * . 1.70 0.75 Tyr 149 . . B. . . . 0.82 −0.06 * * . 1.25 0.84 Ile 150 . . B . . . . −0.03 0.00 * *. 1.00 0.72 Pro 151 . . B . . . . 0.18 0.69 . * . 0.15 0.80 Glu 152 . .B . . . . −0.03 0.04 . * F 0.65 0.89 Thr 153 . . B . . . . 0.48 −0.29 *. F 1.70 1.96 Leu 154 . . . . . . C 0.72 −0.54 * . F 2.50 1.25 Glu 155 .. . . . T C 1.40 −0.57 * . F 3.00 1.25 Pro 156 . . . . T T . 0.76−0.14 * . F 2.60 1.34 Gly 157 . . . . T T . 0.44 0.01 . . F 1.70 1.21Gln 158 . . . . . T C −0.10 −0.19 . . F 1.80 1.01 Pro 159 . . B B . . .−0.18 0.46 . . F −0.15 0.48 Val 160 . . B B . . . −0.84 0.71 . . F −0.450.34 Thr 161 . . B B . . . −1.49 0.86 . . . −0.60 0.11 Val 162 . . B B .. . −1.84 1.10 . . . −0.60 0.05 Ile 163 . . B B . . . −1.84 1.46 . . .−0.60 0.06 Cys 164 . . B B . . . −1.92 1.21 . . . −0.60 0.07 Val 165 . .B B . . . −1.66 1.64 . . . −0.60 0.09 Phe 166 . A B B . . . −2.04 1.50 *. . −0.60 0.13 Asn 167 . A . B T . . −1.19 1.60 * . . −0.20 0.22 Trp 168. A . B T . . −0.30 1.03 * . . −0.20 0.51 Ala 169 . A . B . . C −0.300.39 * . . 0.05 1.02 Phe 170 . A . . T . . 0.34 0.17 * . . 0.10 0.34 Glu171 . A . . T . . 0.83 0.20 * * . 0.10 0.50 Glu 172 . A . . T . . 0.62−0.29 . . F 0.95 0.76 Cys 173 . A . . . . C 0.61 −0.36 . . F 1.00 1.36Pro 174 . A . . . . C 0.50 −0.76 . . F 1.40 1.05 Pro 175 . . . . . T C0.90 0.03 * . F 0.85 0.53 Pro 176 . . . . T T . 0.61 0.41 . . F 1.001.32 Ser 177 . . . . T T . 0.30 0.76 . . F 0.75 0.90 Phe 178 . . . . T T. 0.62 0.81 . . . 0.50 0.84 Ser 179 . . B B . . . 0.24 0.81 . . . −0.400.54 Trp 180 . . B B . . . −0.13 0.89 . . . −0.50 0.40 Thr 181 . . B B .. . −0.73 1.00 . . . −0.60 0.47 Gly 182 . . . B . . C −0.73 0.90 . . .−0.40 0.29 Ala 183 . . . . . . C −0.33 0.90 . . . −0.20 0.37 Ala 184 . .. . . . C −0.03 0.37 . . . 0.10 0.34 Leu 185 . . . . . . C −0.09 0.29 .. F 0.53 0.60 Ser 186 . . . . . . C −0.09 0.29 * . F 0.81 0.59 Ser 187 .. . . T T . 0.30 0.27 * . F 1.49 0.84 Gln 188 . . . . T T . 0.68 −0.23 .. F 2.52 2.04 Gly 189 . . . . T T . 0.96 −0.49 * . F 2.80 2.35 Thr 190 .. . . . T C 1.46 −0.39 * . F 2.32 2.53 Lys 191 . . . . . T C 1.46 −0.29. . F 2.04 2.11 Pro 192 . . B . . T . 1.72 −0.30 * . F 1.56 2.86 Thr 193. . B . . T . 1.02 −0.23 * . F 1.28 2.69 Thr 194 . . B . . T . 1.07 0.07. . F 0.40 1.17 Ser 195 . . B B . . . 0.52 0.46 * . F −0.30 1.01 His 196. . B B . . . −0.33 0.67 * . . −0.60 0.52 Phe 197 . . B B . . . −0.420.87 . . . −0.60 0.30 Ser 198 . . B B . . . −0.81 0.77 . . . −0.60 0.30Val 199 . . B B . . . −0.81 1.17 . . . −0.60 0.19 Leu 200 . . B B . . .−0.72 1.16 * * . −0.60 0.31 Ser 201 . . . B T . . −0.58 0.80 * * . −0.200.36 Phe 202 . . . B . . C −0.09 0.41 * * . −0.06 0.96 Thr 203 . . . . .T C 0.21 0.20 * * F 1.28 1.80 Pro 204 . . . . . T C 1.07 −0.09 * * F2.22 2.32 Arg 205 . . . . . T C 1.84 −0.47 . * F 2.56 4.48 Pro 206 . . .. T T . 2.14 −0.76 . * F 3.40 4.22 Gln 207 . . . . T . . 2.53 −0.84 * *F 2.86 4.39 Asp 208 . . . . T . . 2.84 −0.79 * * F 2.52 3.24 His 209 . .. . T . . 2.24 −0.79 * * F 2.18 3.50 Asn 210 . . . . T T . 1.82−0.53 * * F 2.04 1.67 Thr 211 . . . . T T . 1.37 −0.44 . . F 1.40 1.44Asp 212 . . . . T T . 1.33 0.13 . * F 0.65 0.57 Leu 213 . . B . . T .0.48 0.13 * * . 0.10 0.48 Thr 214 . . B B . . . 0.51 0.37 . * . −0.300.25 Cys 215 . . B B . . . −0.19 −0.11 . * . 0.49 0.25 His 216 . . B B .. . −0.18 0.67 * * . −0.22 0.26 Val 217 . . B B . . . −0.07 0.37 * * .0.27 0.24 Asp 218 . . B B . . . 0.79 −0.11 * * . 1.06 0.88 Phe 219 . . B. . . . 0.76 −0.69 * . . 1.90 1.29 Ser 220 . . . . T T . 0.57 −0.76 * .F 2.46 1.72 Arg 221 . . . . T T . 0.30 −0.76 * . F 2.12 0.77 Lys 222 . .. . T T . 0.30 −0.37 * * F 1.78 1.18 Gly 223 . . . . T T . 0.30 −0.51 *. F 1.74 0.66 Val 224 . . . B . . C 1.11 −0.50 * . F 0.95 0.58 Ser 225 .. B B . . . 1.10 −0.50 * . . 0.60 0.57 Val 226 . . B B . . . 0.13−0.01 * * . 0.30 0.83 Gln 227 . . B B . . . 0.20 0.20 * * F −0.15 0.83Arg 228 . . B B . . . −0.27 −0.44 * * F 0.60 1.21 Thr 229 . . B B . . .0.70 −0.14 * * F 0.60 1.35 Val 230 . . B B . . . 0.14 −0.79 * * . 0.751.52 Arg 231 . . B B . . . 0.41 −0.54 * * . 0.60 0.58 Leu 232 . . B B .. . 0.17 −0.04 * * . 0.30 0.40 Arg 233 . . B B . . . −0.53 0.23 * * .−0.30 0.85 Val 234 . . B B . . . −0.43 0.09 * * . −0.30 0.44 Ala 235 . .B B . . . 0.53 0.51 * * . −0.60 0.82 Tyr 236 . . B B . . . 0.42−0.17 * * . 0.30 0.82 Ala 237 . . B . . T . 0.42 −0.17 * * . 0.85 1.85Pro 238 . . B . . T . −0.54 −0.13 * * . 0.85 1.51 Arg 239 . . B . . T .−0.58 0.01 * . F 0.25 0.72 Asp 240 . . B . . T . −0.29 −0.06 * . F 0.850.50 Leu 241 . . B B . . . −0.93 −0.17 * . . 0.30 0.43 Val 242 . . B B .. . −0.64 0.09 * . . −0.30 0.15 Ile 243 . . B B . . . −0.32 0.47 * . .−0.26 0.12 Ser 244 . . B B . . . −0.43 0.47 * . . 0.08 0.29 Ile 245 . .B B . . . −0.43 −0.21 * . . 1.32 0.66 Ser 246 . . B . . T . 0.07 −0.46 *. F 2.36 1.52 Arg 247 . . . . T T . 0.71 −0.66 * . F 3.40 1.63 Asp 248 .. . . T T . 1.01 −0.61 * . F 3.06 3.61 Asn 249 . . . . . T C 0.50−0.80 * . F 2.52 2.72 Thr 250 . . . . . . C 1.39 −0.50 * . F 1.98 1.14Pro 251 . . . . . . C 1.48 −0.50 . . F 1.64 1.19 Ala 252 . . . . T . .1.37 −0.07 * . F 1.20 1.14 Leu 253 . . B . . . . 1.16 −0.07 . . F 1.141.37 Glu 254 . . B . . . . 1.16 −0.13 . * F 1.48 1.37 Pro 255 . . B . .. . 1.12 −0.16 . * F 1.82 2.35 Gln 256 . . . . . . C 1.33 −0.23 . * F2.36 2.82 Pro 257 . . . . T T . 1.07 −0.51 . * F 3.40 2.62 Gln 258 . . .. T T . 1.67 0.13 . * F 2.16 1.26 Gly 259 . . . . T T . 1.42 0.13 . * F1.82 1.12 Asn 260 . . . . . T C 0.82 0.49 . * F 0.98 1.14 Val 261 . . B. . . . 0.82 0.74 . * F 0.09 0.54 Pro 262 . A B . . . . 0.44 0.34 . . .−0.30 0.95 Tyr 263 . A B . . . . 0.44 0.41 . . . −0.60 0.59 Leu 264 . AB . . . . 0.83 0.41 . . . −0.17 1.39 Glu 265 . A B . . . . 0.49 −0.23 .. . 1.01 1.79 Ala 266 . A B . . . . 1.34 −0.23 . . F 1.44 1.13 Gln 267 .. B . . T . 0.86 −0.59 . . F 2.42 2.38 Lys 268 . . . . T T . 0.29−0.49 * . F 2.80 1.19 Gly 269 . . . . T T . 1.21 0.20 * . F 1.77 0.97Gln 270 . . B . . T . 0.40 −0.30 * . F 1.84 1.10 Phe 271 . A B . . . .0.18 −0.01 * . . 0.86 0.45 Leu 272 . A B . . . . −0.49 0.67 * . . −0.320.38 Arg 273 . A B . . . . −1.12 0.81 * . . −0.60 0.12 Leu 274 . A B . .. . −1.37 0.91 * . . −0.60 0.14 Leu 275 . A B . . . . −1.37 0.63 * . .−0.60 0.17 Cys 276 . A B . . . . −0.97 −0.06 * * . 0.54 0.14 Ala 277 . A. . T . . −0.16 0.33 * * . 0.58 0.23 Ala 278 . . . . T T . −0.480.04 * * F 1.37 0.49 Asp 279 . . . . T T . 0.12 −0.21 . . F 2.36 1.40Ser 280 . . . . . T C 0.34 −0.36 . . F 2.40 2.15 Gln 281 . . . . . T C0.70 −0.36 . . F 2.16 2.15 Pro 282 . . . . . T C 0.48 −0.37 . . F 1.921.85 Pro 283 . . . . T T . 0.77 0.31 . * F 1.28 1.14 Ala 284 . . . . T T. 0.48 0.31 . . F 0.89 0.88 Thr 285 . . B . . T . −0.08 0.83 . . . −0.200.60 Leu 286 . . B B . . . −0.89 1.04 * * . −0.60 0.29 Ser 287 . . B B .. . −0.68 1.30 . . . −0.60 0.24 Trp 288 . . B B . . . −0.47 1.20 . * .−0.60 0.28 Val 289 . . B B . . . 0.23 1.11 . * . −0.60 0.55 Leu 290 . .B B . . . −0.31 0.43 . * . −0.60 0.80 Gln 291 . . B B . . . −0.31 0.69. * F −0.45 0.57 Asn 292 . . B B . . . −0.31 0.46 . * F −0.45 0.63 Arg293 . . B B . . . −0.32 0.20 . * F 0.00 1.03 Val 294 . . B B . . . 0.23−0.10 . . F 0.45 0.79 Leu 295 . . B . . T . 1.01 −0.11 * * F 0.85 0.66Ser 296 . . B . . T . 0.80 −0.01 * . F 0.85 0.46 Ser 297 . . . . T T .0.51 0.41 * . F 0.35 0.96 Ser 298 . . . . . T C 0.06 0.69 * . F 0.301.22 His 299 . . . . . T C 0.70 0.43 * . F 0.15 0.90 Pro 300 . . . . T T. 1.62 0.47 * . . 0.35 1.04 Trp 301 . . . . T T . 1.71 0.09 * . . 0.651.52 Gly 302 . . . . . T C 1.20 0.13 * . F 0.60 1.73 Pro 303 . . . . . .C 1.16 0.31 . . F 0.25 0.92 Arg 304 . . . . . T C 0.38 0.31 . . F 0.450.87 Pro 305 . . . . . T C 0.59 0.09 * . F 0.45 0.72 Leu 306 . . B . . T. 0.07 −0.34 * . . 0.70 0.81 Gly 307 . . B . . T . 0.20 −0.09 * . . 0.700.34 Leu 308 . . B . . . . 0.07 0.34 * . . −0.10 0.34 Glu 309 . . B . .. . −0.90 0.34 * . . −0.10 0.41 Leu 310 . . B . . . . −0.64 0.30 . . .−0.10 0.31 Pro 311 . . B . . . . −0.42 −0.13 . . F 0.65 0.74 Gly 312 . .B . . . . −0.42 −0.31 . . F 0.65 0.43 Val 313 . . B . . . . 0.39 0.11 *. F 0.05 0.52 Lys 314 . . B . . . . 0.09 −0.57 . . F 1.29 0.56 Ala 315 .. B . . . . 0.56 −0.61 * * F 1.63 0.76 Gly 316 . . . . T . . 0.88−0.61 * * F 2.52 1.02 Asp 317 . . . . T T . 0.98 −1.26 * * F 2.91 0.99Ser 318 . . . . T T . 1.52 −0.50 * * F 3.40 1.54 Gly 319 . . . . T T .0.81 −0.51 * * F 3.06 2.25 Arg 320 . . B . . T . 1.51 −0.37 * * F 1.870.72 Tyr 321 . . B B . . . 1.27 −0.37 * * F 1.28 1.05 Thr 322 . . B B .. . 1.27 −0.26 * * . 0.79 1.08 Cys 323 . . B B . . . 1.57 −0.69 * * .0.94 0.95 Arg 324 . . B B . . . 2.02 −0.29 * * . 0.98 0.98 Ala 325 . . B. . . . 1.10 −1.04 * * F 2.12 1.33 Glu 326 . . B . . . . 1.00 −0.84 * *F 2.46 2.04 Asn 327 . . . . T T . 1.01 −0.99 * * F 3.40 1.03 Arg 328 . .. . T T . 1.68 −0.60 * * F 3.06 1.37 Leu 329 . . . . T T . 1.57−0.70 * * F 2.72 1.37 Gly 330 . . . . T T . 2.27 −0.30 * * F 2.08 1.47Ser 331 . . . . . . C 1.68 −0.70 * * F 1.64 1.47 Gln 332 . A B . . . .0.87 −0.20 * * F 0.60 1.80 Gln 333 . A B . . . . 0.76 −0.20 . * F 0.601.50 Arg 334 . A B . . . . 0.76 −0.63 * . F 0.90 1.87 Ala 335 . A B . .. . 0.80 −0.33 * . F 0.45 0.89 Leu 336 . A B . . . . 0.24 −0.34 * * .0.30 0.69 Asp 337 . A B . . . . 0.24 −0.10 * * . 0.30 0.26 Leu 338 . A B. . . . 0.00 0.30 * * . −0.30 0.45 Ser 339 . . B . . . . −0.32 0.56 * *. −0.40 0.85 Val 340 . . B . . . . 0.06 0.30 * * . −0.10 0.79 Gln 341 .. B . . . . 0.87 0.73 . * . −0.25 1.48 Tyr 342 . . B . . . . 0.87 0.04. * . 0.25 1.91 Pro 343 . . . . . T C 0.87 0.06 * * F 1.00 4.14 Pro 344. . . . . T C 1.28 0.10 * * F 1.20 1.97 Glu 345 . . . . T T . 1.28−0.30 * * F 2.20 2.47 Asn 346 . . B . . T . 0.68 −0.41 * * F 2.00 1.18Leu 347 . . B B . . . 0.07 −0.23 * * . 1.10 0.76 Arg 348 . . B B . . .−0.02 −0.01 * * . 0.90 0.32 Val 349 . . B B . . . 0.19 0.37 * * . 0.100.27 Met 350 . . B B . . . −0.40 0.37 * * . −0.10 0.57 Val 351 . . B B .. . −0.40 0.19 * * . −0.30 0.29 Ser 352 . . B . . . . 0.52 0.59 * * .−0.40 0.63 Gln 353 . . B . . . . 0.10 −0.06 * * F 0.80 1.26 Ala 354 . .B . . . . 0.10 −0.19 * . F 0.80 2.44 Asn 355 . . B . . . . −0.11 −0.19 *. F 0.80 1.35 Arg 356 . . B . . . . 0.74 0.11 * . F 0.05 0.64 Thr 357 .. B . . . . 1.04 −0.29 * . F 0.80 1.10 Val 358 . . B . . . . 0.23−0.39 * . . 0.65 1.10 Leu 359 . . B . . . . 0.48 −0.10 * . . 0.50 0.47Glu 360 . . B . . . . 0.48 0.33 * . . 0.03 0.32 Asn 361 . . B . . . .0.02 0.24 * . F 0.31 0.69 Leu 362 . . . . T T . 0.02 0.03 * . F 1.040.83 Gly 363 . . . . T T . 0.58 −0.17 * . F 1.77 0.69 Asn 364 . . . . TT . 0.58 0.21 * . F 1.30 0.58 Gly 365 . . . . . T C 0.37 0.50 * . F 0.670.58 Thr 366 . . . . . . C −0.49 0.24 . . F 0.64 0.90 Ser 367 . . B . .. . −0.49 0.46 . . F 0.01 0.42 Leu 368 . . B . . . . −0.14 0.74 . . .−0.27 0.35 Pro 369 . . B . . . . −0.49 0.31 . . . −0.10 0.42 Val 370 . .B . . . . −0.14 0.26 . . . −0.10 0.31 Leu 371 . . B . . . . −0.13 0.27 .. F 0.05 0.64 Glu 372 . . B . . . . −0.64 −0.03 . . F 0.65 0.56 Gly 373. . . . T T . −0.50 0.23 . . F 0.65 0.62 Gln 374 . . . . T T . −1.100.16 . . F 0.65 0.40 Ser 375 . . . . T T . −1.10 0.16 . . F 0.65 0.19Leu 376 . . B . . T . −0.96 0.80 . . . −0.20 0.14 Cys 377 . . B B . . .−1.81 0.94 . . . −0.60 0.04 Leu 378 . . B B . . . −1.78 1.19 . . . −0.600.02 Val 379 . . B B . . . −1.81 1.29 . . . −0.60 0.04 Cys 380 . . B B .. . −1.81 1.10 . . . −0.60 0.11 Val 381 . . B B . . . −1.30 0.91 . . .−0.60 0.18 Thr 382 . . B B . . . −0.84 0.61 . . . −0.60 0.32 His 383 . .. . T T . −0.24 0.40 . . F 0.89 0.93 Ser 384 . . . . . T C 0.02 0.26 * *F 1.08 1.93 Ser 385 . . . . . T C 0.80 0.11 * * F 1.32 1.35 Pro 386 . .. . . T C 0.84 −0.37 . * F 2.16 1.95 Pro 387 . . . . T . . 0.86 −0.19. * F 2.40 1.20 Ala 388 . . . . T . . 0.60 −0.19 . * F 2.16 1.20 Arg 389. . B B . . . 0.59 0.34 . * . 0.42 0.82 Leu 390 . . B B . . . 0.89 0.40. * . 0.18 0.76 Ser 391 . . B B . . . 1.21 0.37 . * . 0.09 1.30 Trp 392. . B B . . . 1.08 −0.13 . * . 0.45 1.30 Thr 393 . . B B . . . 1.67 0.30. * F 0.00 1.56 Gln 394 . . B . . T . 0.70 0.01 . * F 0.40 2.02 Arg 395. . B . . T . 0.70 0.27 . * F 0.40 1.43 Gly 396 . . . . T T . 0.70 0.04. * F 0.65 0.82 Gln 397 . . B . . T . 0.78 −0.06 . * F 0.85 0.63 Val 398. . B . . . . 0.79 −0.03 . . F 0.65 0.50 Leu 399 . . B . . . . 0.79 0.36. . F 0.05 0.67 Ser 400 . . B . . T . 0.47 0.33 . * F 0.55 0.67 Pro 401. . . . T T . 0.51 0.36 . . F 1.40 1.41 Ser 402 . . . . T T . 0.51 0.10. . F 1.70 2.28 Gln 403 . . . . . T C 1.16 −0.59 . . F 2.70 2.85 Pro 404. . . . T . . 1.62 −0.54 . . F 3.00 2.85 Ser 405 . . . . . . C 1.07−0.54 . . F 2.50 2.10 Asp 406 . . . . . T C 0.47 −0.29 . . F 1.95 0.90Pro 407 . . B . . T . 0.77 0.00 . . F 1.45 0.48 Gly 408 . . B . . T .−0.04 −0.43 . . F 1.15 0.62 Val 409 . . B . . T . −0.04 −0.13 * . . 0.700.31 Leu 410 . . B . . . . 0.37 0.30 * . . −0.10 0.31 Glu 411 . . B . .. . −0.49 −0.13 * . . 0.50 0.61 Leu 412 . . B B . . . −0.28 0.09 * * .−0.30 0.61 Pro 413 . . B B . . . −0.79 −0.16 * * F 0.60 1.27 Arg 414 . AB B . . . 0.07 −0.20 * * . 0.30 0.55 Val 415 . A B B . . . 0.84 −0.20. * . 0.45 1.15 Gln 416 . A B B . . . 0.84 −0.39 . * . 0.45 1.01 Val 417. A B B . . . 1.31 −0.81 . * . 0.60 0.89 Glu 418 . A B B . . . 1.52−0.39 . * . 0.45 1.19 His 419 . A . . . . C 0.71 −1.03 . * F 1.10 1.19Glu 420 . A . . T . . 1.26 −0.64 . * F 1.30 1.39 Gly 421 . A . . T . .0.59 −0.80 . * F 1.30 1.16 Glu 422 . A . . T . . 1.41 −0.23 * * F 0.850.46 Phe 423 . A . . T . . 0.82 −0.23 * * . 0.70 0.36 Thr 424 A A . . .. . 0.97 0.27 . * . −0.30 0.37 Cys 425 . A . . T . . 0.93 −0.16 * * .0.70 0.41 His 426 . A B . . . . 1.07 0.34 * * . −0.30 0.65 Ala 427 . A .. T . . 0.26 −0.01 * * . 0.95 0.70 Arg 428 . A . . . . C 0.61 0.19 * * .0.55 1.07 His 429 . . . . . T C 0.62 0.04 . * . 1.05 0.78 Pro 430 . . .. T T . 1.29 −0.07 . * . 2.25 1.03 Leu 431 . . . . T T . 1.29 −0.17 . *F 2.50 0.91 Gly 432 . . . . T T . 1.02 0.33 . * F 1.65 0.91 Ser 433 . .B B . . . 0.61 0.47 . * F 0.30 0.44 Gln 434 . . B B . . . −0.17 0.43 . .F 0.05 0.71 His 435 . . B B . . . −0.26 0.43 . * . −0.35 0.59 Val 436 .. B B . . . −0.26 0.39 . * . −0.30 0.59 Ser 437 . . B B . . . −0.21 0.69. * . −0.60 0.28 Leu 438 . . B B . . . −0.77 0.67 . * . −0.60 0.28 Ser439 . . B B . . . −0.80 0.81 . * . −0.60 0.28 Leu 440 . . B B . . .−1.01 0.67 . * . −0.60 0.28 Ser 441 . . B B . . . −0.46 1.04 . * . −0.600.54 Val 442 . . B B . . . −0.37 0.74 . * . −0.60 0.54 His 443 . . B B .. . 0.49 0.79 * * . −0.45 1.01 Tyr 444 . . B B . . . −0.02 0.10 * * .−0.15 1.50 Ser 445 . . B . . T . −0.02 0.40 . * . 0.25 1.67 Pro 446 . .B . . T . −0.07 0.44 * * F 0.10 1.01 Lys 447 . . . . T T . 0.58 0.37 * *F 0.65 0.64 Leu 448 . . . . T T . 0.31 0.04 * . F 0.65 0.74 Leu 449 . .B . . . . −0.11 0.04 . . F 0.05 0.64 Gly 450 . . . . . T C −0.11 0.19 .. F 0.45 0.17 Pro 451 . . . . . T C −0.19 0.57 . . F 0.15 0.28 Ser 452 .. . . . T C −0.23 0.80 . . F 0.15 0.36 Cys 453 . . . . . T C −0.01 0.11. * . 0.30 0.62 Ser 454 . A . . . . C 0.80 0.19 . * . −0.10 0.41 Trp 455. A B . . . . 0.80 −0.24 . . . 0.30 0.52 Glu 456 . A B . . . . 0.20−0.20 . . . 0.30 0.97 Ala 457 . A . . T . . 0.47 −0.09 . . . 0.70 0.60Glu 458 . A . . T . . 0.47 0.03 . . F 0.10 0.77 Gly 459 . A . . T . .0.47 −0.31 . . . 0.70 0.24 Leu 460 . A . . T . . 0.09 0.07 . . . 0.100.32 His 461 . A . . T . . −0.21 0.14 . . . 0.10 0.10 Cys 462 . . . . TT . 0.08 0.53 . . . 0.20 0.13 Ser 463 . . . . T T . 0.08 0.49 . . . 0.200.22 Cys 464 . . . . T T . −0.17 0.20 . . . 0.50 0.27 Ser 465 . . . . TT . 0.34 0.20 . * F 0.65 0.52 Ser 466 . . . . T . . 0.17 0.01 . . F 0.450.52 Gln 467 . . . . T . . 0.24 0.06 . . F 0.60 1.49 Ala 468 . . . . . .C 0.33 −0.01 . . F 1.24 1.13 Ser 469 . . . . . . C 0.70 0.03 . . F 0.881.30 Pro 470 . . . . . . C 0.19 0.03 . * F 1.12 1.00 Ala 471 . . . . . TC 0.60 0.31 . * F 1.41 0.82 Pro 472 . . . . . T C 0.31 −0.19 * * F 2.401.20 Ser 473 . . . . . T C 0.61 0.34 * * F 1.41 0.82 Leu 474 . . B . . T. 0.10 0.83 * * . 0.52 0.85 Arg 475 . . B B . . . −0.03 1.01 * * . −0.120.45 Trp 476 . . B B . . . 0.56 1.01 . * . −0.36 0.33 Trp 477 . A . B .. C 0.77 0.63 . * . −0.40 0.70 Leu 478 . A . B . . C 0.26 −0.06 . * .0.50 0.62 Gly 479 . A . B . . C 0.26 0.63 . * . −0.40 0.49 Glu 480 . A B. . . . 0.14 0.40 . * F −0.15 0.38 Glu 481 . A B . . . . 0.09 −0.51 . .F 0.75 0.80 Leu 482 . A . . . . C 0.38 −0.77 * . F 0.95 0.80 Leu 483 . A. . . . C 0.89 −0.80 * . F 1.25 0.74 Glu 484 . A . . T . . 0.93 −0.41 .. F 1.45 0.58 Gly 485 . A . . T . . 0.93 −0.03 . . F 1.75 0.94 Asn 486 .. . . T T . 0.93 −0.31 . . F 2.60 1.97 Ser 487 . . . . . T C 1.44 −1.00. . F 3.00 1.89 Ser 488 . . . . . T C 1.56 −0.61 . * F 2.70 2.57 Gln 489. . . . . T C 1.56 −0.26 . * F 2.10 1.38 Asp 490 . A . . . . C 1.04−0.66 * . F 1.70 1.79 Ser 491 . A B . . . . 0.73 −0.40 * . F 0.75 0.99Phe 492 . A B . . . . 0.82 −0.30 . * . 0.30 0.82 Glu 493 . A B . . . .0.82 −0.27 . . . 0.43 0.76 Val 494 . A B . . . . 0.52 0.11 * . F 0.110.76 Thr 495 . . B . . T . −0.07 0.11 . . F 0.79 1.18 Pro 496 . . . . .T C −0.11 −0.17 . * F 1.57 0.69 Ser 497 . . . . T T . 0.38 0.26 . . F1.30 0.92 Ser 498 . . . . . T C 0.09 0.04 . . F 0.97 0.98 Ala 499 . . .. . . C 0.36 0.47 . . F 0.34 0.67 Gly 500 . . . . . T C 0.67 0.54 . . F0.41 0.50 Pro 501 . . . . T T . 0.58 0.56 . . F 0.48 0.61 Trp 502 . . .. . T C 0.58 0.56 * . F 0.15 0.80 Ala 503 . . B . . T . 0.07 0.44 * . F0.10 1.09 Asn 504 . . B . . T . 0.36 0.70 . . F −0.05 0.58 Ser 505 . . B. . T . −0.11 0.66 . * F −0.05 0.74 Ser 506 . . B . . T . 0.07 0.43 . *F −0.05 0.60 Leu 507 . . B . . T . 0.01 0.43 . * . −0.20 0.51 Ser 508 .. B . . . . 0.26 0.46 . * . −0.40 0.38 Leu 509 . . B . . T . −0.56 0.50. * . −0.20 0.28 His 510 . . B . . T . −0.56 0.80 . * . −0.20 0.28 Gly511 . . . . T T . −0.56 0.50 . * . 0.20 0.28 Gly 512 . . . . . T C −0.090.50 . * F 0.15 0.45 Leu 513 . . . . . . C −0.60 0.24 * * F 0.39 0.33Ser 514 . . . . . T C 0.32 0.43 * * F 0.43 0.27 Ser 515 . . . . T T .−0.46 0.00 . * F 1.67 0.54 Gly 516 . . B . . T . 0.00 0.26 . * F 0.810.54 Leu 517 . . B . . T . −0.32 −0.43 * * . 1.40 0.79 Arg 518 . A B . .. . 0.49 −0.24 * * . 0.86 0.32 Leu 519 . A B . . . . 0.20 −0.63 * * .1.02 0.56 Arg 520 . A B . . . . 0.21 −0.56 * * . 0.88 0.68 Cys 521 . A B. . . . 0.56 −0.33 * * . 0.44 0.37 Glu 522 . A B . . . . 0.51 0.07 . * .−0.30 0.71 Ala 523 . A . . T . . 0.37 0.03 * * . 0.10 0.27 Trp 524 . A .. T . . 0.83 0.53 * * . −0.20 0.68 Asn 525 . . B . . T . 0.13 0.39 . . .0.10 0.39 Val 526 . . B . . T . 0.80 0.89 . . . −0.20 0.39 His 527 . . .. . T C 0.50 0.79 . . . 0.00 0.64 Gly 528 . . . . . T C 0.74 0.26 . . .0.30 0.54 Ala 529 . . . . . . C 0.73 0.29 . . F 0.25 0.72 Gln 530 . . .. . T C −0.16 0.03 . . F 0.45 0.71 Ser 531 . . . . . T C −0.11 0.21 . .F 0.45 0.50 Gly 532 . . B . . T . −0.08 0.47 . . F −0.05 0.41 Ser 533 .. B . . T . −0.54 0.37 . . F 0.25 0.41 Ile 534 . . B . . . . −0.17 0.66. . . −0.40 0.25 Leu 535 . . B . . . . −0.17 0.70 * . . −0.06 0.39 Gln536 . . B . . . . 0.18 0.27 * . . 0.58 0.49 Leu 537 . . B . . T . 0.57−0.11 * . . 1.87 1.39 Pro 538 . . B . . T . 0.52 −0.80 * . F 2.66 3.38Asp 539 . . . . T T . 0.60 −1.06 . . F 3.40 1.93 Lys 540 . . . . T T .0.52 −0.77 . . F 3.06 1.93 Lys 541 . . . B T . . 0.22 −0.77 . . F 2.170.88 Gly 542 . . B B . . . 0.72 −0.81 . . F 1.43 0.70 Leu 543 . . B B .. . 0.34 −0.33 . . F 0.79 0.51 Ile 544 . . B B . . . −0.36 0.17 . . .−0.30 0.26 Ser 545 . . B B . . . −0.70 0.96 . . . −0.60 0.22 Thr 546 . .B B . . . −0.74 0.91 * . . −0.60 0.36 Ala 547 . . B B . . . −0.74 0.63 *. . −0.60 0.84 Phe 548 . . B . . T . −0.52 0.37 . . F 0.25 0.62 Ser 549. . . . . T C −0.33 0.49 * . F 0.15 0.43 Asn 550 . . . . . T C −0.840.79 . . F 0.15 0.37 Gly 551 . . . . . T C −0.88 0.97 . . F 0.15 0.35Ala 552 . . . B . . C −1.18 0.61 . . . −0.40 0.26 Phe 553 . . B B . . .−0.82 0.91 . . . −0.60 0.11 Leu 554 . . B B . . . −1.41 0.94 . . . −0.600.11 Gly 555 . . B B . . . −1.72 1.20 . . . −0.60 0.08 Ile 556 . . B B .. . −1.97 1.19 . . . −0.60 0.13 Gly 557 . . B B . . . −2.19 0.90 . . .−0.60 0.16 Ile 558 . A B . . . . −2.30 0.90 . . . −0.60 0.13 Thr 559 . AB . . . . −2.19 1.16 . . . −0.60 0.16 Ala 560 . A B . . . . −2.66 1.26 .. . −0.60 0.14 Leu 561 . A B . . . . −2.43 1.51 . . . −0.60 0.16 Leu 562. A B . . . . −2.90 1.40 . . . −0.60 0.06 Phe 563 . A B . . . . −2.601.60 . . . −0.60 0.05 Leu 564 . A B . . . . −3.10 1.60 . . . −0.60 0.06Cys 565 A A . . . . . −3.40 1.60 . . . −0.60 0.06 Leu 566 A A . . . . .−3.48 1.60 . . . −0.60 0.05 Ala 567 A A . . . . . −3.27 1.50 * . . −0.600.04 Leu 568 A A . . . . . −2.52 1.43 * . . −0.60 0.08 Ile 569 . A B . .. . −2.60 0.86 * . . −0.60 0.19 Ile 570 . A B . . . . −2.74 0.86 * . .−0.60 0.13 Met 571 . A B . . . . −2.14 1.04 * . . −0.60 0.13 Lys 572 . AB . . . . −1.51 0.79 * . . −0.26 0.28 Ile 573 . A B . . . . −0.59 0.10 .. . 0.38 0.81 Leu 574 . A B . . . . 0.41 −0.59 . * . 1.77 1.61 Pro 575 .. . . . T C 0.99 −1.20 . . F 2.86 1.57 Lys 576 . . . . T T . 1.59 −0.71. * F 3.40 3.24 Arg 577 . . . . T T . 1.23 −1.00 . * F 3.06 6.80 Arg 578. . . . T T . 2.12 −1.20 . . F 2.72 6.34 Thr 579 . . . . T . . 2.62−1.63 . . F 2.18 5.49 Gln 580 . . B . . . . 2.62 −1.14 * . F 1.44 4.05Thr 581 . . B . . . . 2.69 −0.71 * . F 1.10 3.20 Glu 582 . . B . . . .2.37 −0.71 * * F 1.10 4.34 Thr 583 . . B . . T . 2.37 −0.77 . * F 1.303.87 Pro 584 . . . . . T C 1.98 −1.17 . * F 1.50 5.26 Arg 585 . . . . .T C 1.68 −0.87 * * F 1.84 2.63 Pro 586 . . . . T T . 2.10 −0.49 * * F2.08 2.44 Arg 587 . . . . T . . 2.07 −0.97 * * F 2.52 3.09 Phe 588 . . .. T . . 2.08 −0.90 * * F 2.86 2.15 Ser 589 . . . . T T . 1.98 −0.51 . *F 3.40 1.86 Arg 590 . . B . . T . 0.98 −0.46 * * F 2.36 1.37 His 591 . .B . . T . 0.38 0.23 * . F 1.42 1.11 Ser 592 . . B . . T . 0.27 0.13 * .F 0.93 0.68 Thr 593 . . B B . . . 0.72 −0.26 * . . 0.64 0.58 Ile 594 . .B B . . . 0.13 0.50 * . . −0.60 0.67 Leu 595 . . B B . . . 0.02 0.69 . *. −0.60 0.35 Asp 596 . . B B . . . −0.80 0.70 * . . −0.60 0.39 Tyr 597 .. B B . . . −1.36 0.86 * . . −0.60 0.41 Ile 598 . . B B . . . −1.260.81 * . . −0.60 0.37 Asn 599 . . B B . . . −0.68 0.56 * . . −0.60 0.34Val 600 . . B B . . . −0.46 1.04 * . . −0.60 0.32 Val 601 . . B B . . .−0.80 0.79 * . . −0.60 0.46 Pro 602 . . B . . . . −0.77 0.53 * . F −0.250.28 Thr 603 . . B . . T . −0.69 0.56 * . F −0.05 0.59 Ala 604 . . B . .T . −1.28 0.60 * . F −0.05 0.65 Gly 605 . . B . . T . −0.42 0.46 * . F−0.05 0.43 Pro 606 . . B . . T . 0.48 0.43 * . F 0.21 0.51 Leu 607 . . B. . . . 0.80 −0.06 . . F 1.32 1.01 Ala 608 . . B . . . . 1.11 −0.56 . .F 1.88 2.00 Gln 609 . . B . . . . 1.70 −0.59 . . F 2.14 2.08 Lys 610 . .B . . T . 2.09 −0.61 * . F 2.60 4.38 Arg 611 . . B . . T . 1.71 −1.30 .. F 2.34 8.66 Asn 612 . . B . . T . 2.21 −1.30 . . F 2.08 5.05 Gln 613 .. B . . T . 2.59 −1.21 * . F 2.10 3.65 Lys 614 . . B . . . . 2.59−0.79 * . F 1.92 2.88 Ala 615 . . . . . . C 2.24 −0.39 * . F 1.84 2.88Thr 616 . . . . . T C 1.92 −0.40 * . F 2.32 2.23 Pro 617 . . . . T T .2.03 −0.37 * . F 2.80 1.72 Asn 618 . . . . T T . 1.72 −0.37 * . F 2.523.34 Ser 619 . . . . . T C 1.47 −0.39 * * F 2.04 3.34 Pro 620 . . . . T. . 1.24 −0.44 * . F 1.76 3.34 Arg 621 . . . . T . . 1.34 −0.19 * . F1.48 1.71 Thr 622 . . B . . . . 1.34 −0.16 * . F 0.80 1.98 Pro 623 . . B. . . . 1.00 −0.11 * . F 0.80 1.98 Leu 624 . . B . . . . 0.71 −0.11 . *F 0.65 1.00 Pro 625 . . B . . T . 0.71 0.39 . * F 0.25 0.70 Pro 626 . .. . T T . 0.30 0.33 . * F 0.65 0.70 Gly 627 . . . . . T C 0.40 0.29 . .F 0.60 1.14 Ala 628 . . . . . T C 0.61 0.03 . . F 0.94 1.14 Pro 629 . .. . . . C 1.12 −0.40 . . F 1.68 1.27 Ser 630 . . . . . T C 1.38 −0.44 .. F 2.22 1.72 Pro 631 . . . . . T C 1.63 −0.87 . . F 2.86 3.41 Glu 632 .. . . T T . 1.98 −1.37 . . F 3.40 4.41 Ser 633 . . . . T T . 2.57 −1.40. . F 3.06 5.29 Lys 634 . . . . T T . 2.82 −1.39 . . F 2.94 5.93 Lys 635. . . . T T . 3.17 −1.81 . . F 2.82 6.84 Asn 636 . . . . T T . 3.38−1.81 . . F 2.70 10.21 Gln 637 . . . . T T . 3.13 −1.80 * . F 2.58 8.84Lys 638 . . B . . . . 3.43 −1.04 * . F 2.20 6.93 Lys 639 . . B . . . .2.58 −0.64 * . F 1.98 7.46 Gln 640 . . B . . . . 2.32 −0.36 * . F 1.463.55 Tyr 641 . . B . . . . 2.02 −0.33 . . . 1.09 2.75 Gln 642 . . B . .. . 1.32 0.06 . . . 0.27 1.84 Leu 643 . . B . . T . 1.07 0.84 . . .−0.20 0.92 Pro 644 . . B . . T . 1.02 0.87 . . F −0.05 0.91 Ser 645 . .B . . T . 0.81 0.11 . . F 0.59 0.91 Phe 646 . . B . . T . 1.10 0.14 . .F 1.08 1.70 Pro 647 . . . . . . C 0.80 −0.54 . . F 2.32 2.20 Glu 648 . .. . . . C 1.31 −0.59 . . F 2.66 2.20 Pro 649 . . . . T T . 1.21 −0.59 .. F 3.40 3.41 Lys 650 . . . . T T . 1.51 −0.89 . . F 3.06 3.18 Ser 651 .. . . . T C 1.62 −0.91 . . F 2.52 3.18 Ser 652 . . . . . T C 1.62−0.41 * . F 1.88 2.08 Thr 653 . . . . . . C 1.62 −0.41 * . F 1.64 1.61Gln 654 . . . . . . C 1.53 −0.41 * . F 1.60 2.08 Ala 655 . . . . . T C1.49 −0.41 * . F 2.10 2.08 Pro 656 . . . . . T C 1.79 −0.40 . . F 2.402.49 Glu 657 . . . . . T C 1.79 −0.89 . . F 3.00 2.49 Ser 658 . . . . .T C 2.10 −0.90 . . F 2.70 3.31 Gln 659 . A . . . . C 2.10 −1.00 . . F2.00 3.70 Glu 660 . A . . . . C 2.69 −1.43 . . F 1.70 3.70 Ser 661 . A .. . . C 2.09 −1.43 . . F 1.40 4.79 Gln 662 A A . . . . . 2.06 −1.13 . .F 0.90 2.28 Glu 663 A A . . . . . 2.11 −1.03 * . F 0.90 1.79 Glu 664 A A. . . . . 1.52 −0.27 * . F 0.60 2.09 Leu 665 . A B . . . . 1.21 −0.16 .. . 0.45 1.22 His 666 . A B . . . . 0.70 −0.07 . . . 0.45 1.02 Tyr 667 .A B . . . . 0.70 0.61 . . . −0.60 0.48 Ala 668 . A B . . . . 0.00 1.01 *. . −0.60 0.95 Thr 669 . A B . . . . −0.21 1.11 . * . −0.60 0.60 Leu 670. A . . T . . 0.26 1.04 . . . −0.20 0.59 Asn 671 . A B . . . . −0.570.71 * . . −0.60 0.58 Phe 672 . . B B . . . −0.21 0.86 * . . −0.60 0.30Pro 673 . . . B T . . 0.17 0.37 . * F 0.25 0.71 Gly 674 . . . B T . .0.59 0.11 . * F 0.25 0.68 Val 675 . . . B . . C 1.19 −0.29 . * F 0.801.54 Arg 676 . . . . . T C 1.19 −0.64 . * F 1.50 1.54 Pro 677 . . . . .T C 1.30 −1.07 . * F 1.50 2.70 Arg 678 . . . . . T C 1.62 −1.00 . * F1.50 3.68 Pro 679 . . B . . T . 1.37 −1.64 . * F 1.30 3.68 Glu 680 . . .. T . . 2.01 −1.03 * * F 1.84 2.35 Ala 681 . . B . . . . 1.94 −1.03 * *F 1.78 1.86 Arg 682 . . B . . . . 1.81 −1.03 * * . 1.97 2.40 Met 683 . .B . . T . 1.39 −1.03 * * F 2.66 1.37 Pro 684 . . . . T T . 1.60 −0.54. * F 3.40 1.96 Lys 685 . . . . T T . 1.01 −0.64 . * F 3.06 1.74 Gly 686. . . . . T C 1.60 −0.14 * . F 2.22 1.77 Thr 687 . A . . . . C 1.24−0.76 * . F 1.78 1.91 Gln 688 . A B . . . . 1.26 −0.43 . . F 0.94 1.50Ala 689 . A B . . . . 1.47 0.07 . . F 0.00 1.53 Asp 690 . A B . . . .0.57 −0.36 . . . 0.45 1.84 Tyr 691 . A B . . . . 0.96 −0.20 . * . 0.300.79 Ala 692 . A B . . . . 0.57 −0.60 . * . 0.75 1.56 Glu 693 . A B . .. . 0.57 −0.31 . * . 0.30 0.81 Val 694 . A B . . . . 0.77 0.09 . * .−0.30 0.89 Lys 695 . A B . . . . 0.38 −0.24 . * . 0.45 1.13 Phe 696 . AB . . . . 0.23 −0.31 . * . 0.30 0.83 Gln 697 . A B . . . . 0.43 0.11 . *. −0.15 1.44

TABLE XIV First Last First NT 5′ NT 3′ NT 5′ NT 5′ NT of AA AA AA AA ofLast SEQ Total of of of First SEQ of of Sec- AA Gene cDNA ATCC DepositID NT Clone Clone Start AA of ID Sig Sig reted of No. Clone ID No: Z andDate Vector NO: X Seq. Seq. Seq. Codon Signal Pep NO: Y Pep Pep PortionORF 1 HAOAB64 203484 pSport1 11 2609 1 2609 599 599 29 1 30 31 529 Nov.17, 1998 2 HOHCH55 203484 pCMVSport 2.0 12 2499 1 2499 221 221 30 1 2324 494 Nov. 17, 1998 2 HOHCH55 203484 pCMVSport 2.0 23 2522 1 2522 230230 41 1 23 24 469 Nov. 17, 1998 3 HTLEW81 203484 Uni-ZAP XR 13 1339 11339 37 37 31 1 27 28 148 Nov. 17, 1998 4 HARAO44 203484 pBluescript SK−14 1389 1 1389 125 125 32 1 21 22 332 Nov. 17, 1998 6 HDPUW68 203484pCMVSport 3.0 16 1748 1 1748 40 40 34 1 18 19 467 Nov. 17, 1998 7HOHBY69 203484 pCMVSport 2.0 17 4995 1 4995 82 82 35 1 22 23 1189 Nov.17, 1998 7 HOHBY69 203484 pCMVSport 2.0 25 4631 1 4631 84 84 43 1 22 231034 Nov. 17, 1998 8 HCDDP40 203484 Uni-ZAP XR 18 726 1 726 32 32 36 121 22 196 Nov. 17, 1998 9 HTTDB46 203484 Uni-ZAP XR 19 3059 1 3059 55 5537 1 17 18 318 Nov. 17, 1998 9 HTTDB46 203484 Uni-ZAP XR 26 2008 2152008 153 153 44 1 17 18 461 Nov. 17, 1998 5 148 2338 165 2338 201 201149 1 16 17 697 5 HDPCL05 203484 pCMVSport 3.0 15 2295 1 2295 58 58 33 116 17 639 Nov. 17, 1998 5 HDPCL05 203484 pCMVSport 3.0 24 1344 1 1344 5252 42 1 16 17 127 Nov. 17, 1998 10 HUSAQ05 203484 Lambda ZAP II 20 16991 1699 115 115 38 1 19 20 375 Nov. 17, 1998 10 HUSAQ05 203484 Lambda ZAPII 27 1654 1 1654 115 115 45 1 19 20 383 Nov. 17, 1998 11 HOUDJ81 203484Uni-ZAP XR 21 1520 1 1520 26 26 39 1 44 45 364 Nov. 17, 1998 11 HOUDJ81203484 Uni-ZAP XR 28 1508 19 1508 454 454 46 1 30 31 229 Nov. 17, 199812 HPWCM76 203484 Uni-ZAP XR 22 807 1 807 582 582 40 1 23 24 66 Nov. 17,1998

Table XIV summarizes the information corresponding to each “Gene No.”described above. The nucleotide sequence identified as “NT SEQ ID NO:X”was assembled from partially homologous (“overlapping”) sequencesobtained from the “cDNA clone ID” identified in Table XIV and, in somecases, from additional related DNA clones. The overlapping sequenceswere assembled into a single contiguous sequence of high redundancy(usually three to five overlapping sequences at each nucleotideposition), resulting in a final sequence identified as SEQ ID NO:X.

The cDNA Clone ID was deposited on the date and given the correspondingdeposit number listed in “ATCC Deposit No:Z and Date.” Some of thedeposits contain multiple different clones corresponding to the samegene. “Vector” refers to the type of vector contained in the cDNA CloneID.

“Total NT Seq.” refers to the total number of nucleotides in the contigidentified by “Gene No.” The deposited clone may contain all or most ofthese sequences, reflected by the nucleotide position indicated as “5′NT of Clone Seq.” and the “3′ NT of Clone Seq.” of SEQ ID NO:X. Thenucleotide position of SEQ ID NO:X of the putative start codon(methionine) is identified as “5′ NT of Start Codon.” Similarly, thenucleotide position of SEQ ID NO:X of the predicted signal sequence isidentified as “5′ NT of First AA of Signal Pep.”

The translated amino acid sequence, beginning with the methionine, isidentified as “AA SEQ ID NO:Y,” although other reading frames can alsobe easily translated using known molecular biology techniques. Thepolypeptides produced by these alternative open reading frames arespecifically contemplated by the present invention.

The first and last amino acid position of SEQ ID NO:Y of the predictedsignal peptide is identified as “First AA of Sig Pep” and “Last AA ofSig Pep.” The predicted first amino acid position of SEQ ID NO:Y of thesecreted portion is identified as “Predicted First AA of SecretedPortion.” Finally, the amino acid position of SEQ ID NO:Y of the lastamino acid in the open reading frame is identified as “Last AA of ORF.”

SEQ ID NO:X (where X may be any of the polynucleotide sequencesdisclosed in the sequence listing) and the translated SEQ ID NO:Y (whereY may be any of the polypeptide sequences disclosed in the sequencelisting) are sufficiently accurate and otherwise suitable for a varietyof uses well known in the art and described further below. For instance,SEQ ID NO:X is useful for designing nucleic acid hybridization probesthat will detect nucleic acid sequences contained in SEQ ID NO:X or thecDNA contained in the deposited clone. These probes will also hybridizeto nucleic acid molecules in biological samples, thereby enabling avariety of forensic and diagnostic methods of the invention. Similarly,polypeptides identified from SEQ ID NO:Y may be used, for example, togenerate antibodies which bind specifically to proteins containing thepolypeptides and the secreted proteins encoded by the cDNA clonesidentified in Table XIV.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:X and the predicted translated amino acid sequence identified as SEQID NO:Y, but also a sample of plasmid DNA containing a human cDNA of theinvention deposited with the ATCC, as set forth in Table XIV. Thenucleotide sequence of each deposited clone can readily be determined bysequencing the deposited clone in accordance with known methods. Thepredicted amino acid sequence can then be verified from such deposits.Moreover, the amino acid sequence of the protein encoded by a particularclone can also be directly determined by peptide sequencing or byexpressing the protein in a suitable host cell containing the depositedhuman cDNA, collecting the protein, and determining its sequence.

The present invention also relates to the genes corresponding to SEQ IDNO:X, SEQ ID NO:Y, or the deposited clone. The corresponding gene can beisolated in accordance with known methods using the sequence informationdisclosed herein. Such methods include preparing probes or primers fromthe disclosed sequence and identifying or amplifying the correspondinggene from appropriate sources of genomic material.

Also provided in the present invention are allelic variants, orthologs,and/or species homologs. Procedures known in the art can be used toobtain full-length genes, allelic variants, splice variants, full-lengthcoding portions, orthologs, and/or species homologs of genescorresponding to SEQ ID NO:X, SEQ ID NO:Y, or a deposited clone, usinginformation from the sequences disclosed herein or the clones depositedwith the ATCC. For example, allelic variants and/or species homologs maybe isolated and identified by making suitable probes or primers from thesequences provided herein and screening a suitable nucleic acid sourcefor allelic variants and/or the desired homologue.

The polypeptides of the invention can be prepared in any suitablemanner. Such polypeptides include isolated naturally occurringpolypeptides, recombinantly produced polypeptides, syntheticallyproduced polypeptides, or polypeptides produced by a combination ofthese methods. Means for preparing such polypeptides are well understoodin the art.

The polypeptides may be in the form of the secreted protein, includingthe mature form, or may be a part of a larger protein, such as a fusionprotein (see below). It is often advantageous to include an additionalamino acid sequence which contains secretory or leader sequences,pro-sequences, sequences which aid in purification , such as multiplehistidine residues, or an additional sequence for stability duringrecombinant production.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of a polypeptide, including the secretedpolypeptide, can be substantially purified using techniques describedherein or otherwise known in the art, such as, for example, by theone-step method described in Smith and Johnson, Gene 67:31-40 (1988).Polypeptides of the invention also can be purified from natural,synthetic or recombinant sources using techniques described herein orotherwise known in the art, such as, for example, antibodies of theinvention raised against the secreted protein.

The present invention provides a polynucleotide comprising, oralternatively consisting of, the nucleic acid sequence of SEQ ID NO:Xand/or a cDNA contained in ATCC deposit Z. The present invention alsoprovides a polypeptide comprising, or alternatively, consisting of, thepolypeptide sequence of SEQ ID NO:Y and/or a polypeptide encoded by thecDNA contained in ATCC deposit Z. Polynucleotides encoding a polypeptidecomprising, or alternatively consisting of the polypeptide sequence ofSEQ ID NO:Y and/or a polypeptide sequence encoded by the cDNA containedin ATCC deposit Z are also encompassed by the invention.

Signal Sequences

The present invention also encompasses mature forms of the polypeptidehaving the polypeptide sequence of SEQ ID NO:Y and/or the polypeptidesequence encoded by the cDNA in a deposited clone. Polynucleotidesencoding the mature forms (such as, for example, the polynucleotidesequence in SEQ ID NO:X and/or the polynucleotide sequence contained inthe cDNA of a deposited clone) are also encompassed by the invention.According to the signal hypothesis, proteins secreted by mammalian cellshave a signal or secretary leader sequence which is cleaved from themature protein once export of the growing protein chain across the roughendoplasmic reticulum has been initiated. Most mammalian cells and eveninsect cells cleave secreted proteins with the same specificity.However, in some cases, cleavage of a secreted protein is not entirelyuniform, which results in two or more mature species of the protein.Further, it has long been known that cleavage specificity of a secretedprotein is ultimately determined by the primary structure of thecomplete protein, that is, it is inherent in the amino acid sequence ofthe polypeptide.

Methods for predicting whether a protein has a signal sequence, as wellas the cleavage point for that sequence, are available. For instance,the method of McGeoch, Virus Res. 3:271-286 (1985), uses the informationfrom a short N-terminal charged region and a subsequent uncharged regionof the complete (uncleaved) protein. The method of von Heinje, NucleicAcids Res. 14:4683-4690 (1986) uses the information from the residuessurrounding the cleavage site, typically residues −13 to +2, where +1indicates the amino terminus of the secreted protein. The accuracy ofpredicting the cleavage points of known mammalian secretory proteins foreach of these methods is in the range of 75-80%. (von Heinje, supra.)However, the two methods do not always produce the same predictedcleavage point(s) for a given protein.

In the present case, the deduced amino acid sequence of the secretedpolypeptide was analyzed by a computer program called SignalP (HenrikNielsen et al., Protein Engineering 10:1-6 (1997)), which predicts thecellular location of a protein based on the amino acid sequence. As partof this computational prediction of localization, the methods of McGeochand von Heinje are incorporated. The analysis of the amino acidsequences of the secreted proteins described herein by this programprovided the results shown in Table XIV.

As one of ordinary skill would appreciate, however, cleavage sitessometimes vary from organism to organism and cannot be predicted withabsolute certainty. Accordingly, the present invention provides secretedpolypeptides having a sequence shown in SEQ ID NO:Y which have anN-terminus beginning within 5 residues (i.e., + or −5 residues) of thepredicted cleavage point. Similarly, it is also recognized that in somecases, cleavage of the signal sequence from a secreted protein is notentirely uniform, resulting in more than one secreted species. Thesepolypeptides, and the polynucleotides encoding such polypeptides, arecontemplated by the present invention.

Moreover, the signal sequence identified by the above analysis may notnecessarily predict the naturally occurring signal sequence. Forexample, the naturally occurring signal sequence may be further upstreamfrom the predicted signal sequence. However, it is likely that thepredicted signal sequence will be capable of directing the secretedprotein to the ER. Nonetheless, the present invention provides themature protein produced by expression of the polynucleotide sequence ofSEQ ID NO:X and/or the polynucleotide sequence contained in the cDNA ofa deposited clone, in a mammalian cell (e.g., COS cells, as describedbelow). These polypeptides, and the polynucleotides encoding suchpolypeptides, are contemplated by the present invention.

Polynucleotide and Polypeptide Variants

The present invention is directed to variants of the polynucleotidesequence disclosed in SEQ ID NO:X, the complementary strand thereto,and/or the cDNA sequence contained in a deposited clone.

The present invention also encompasses variants of the polypeptidesequence disclosed in SEQ ID NO:Y and/or encoded by a deposited clone.

“Variant” refers to a polynucleotide or polypeptide differing from thepolynucleotide or polypeptide of the present invention, but retainingessential properties thereof Generally, variants are overall closelysimilar, and, in many regions, identical to the polynucleotide orpolypeptide of the present invention.

The present invention is also directed to nucleic acid molecules whichcomprise, or alternatively consist of, a nucleotide sequence which is atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, forexample, the nucleotide coding sequence in SEQ ID NO:X or thecomplementary strand thereto, the nucleotide coding sequence containedin a deposited cDNA clone or the complementary strand thereto, anucleotide sequence encoding the polypeptide of SEQ ID NO:Y, anucleotide sequence encoding the polypeptide encoded by the cDNAcontained in a deposited clone, and/or polynucleotide fragments of anyof these nucleic acid molecules (e.g., those fragments describedherein). Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions or lower stringency conditionsare also encompassed by the invention, as are polypeptides encoded bythese polynucleotides.

The present invention is also directed to polypeptides which comprise,or alternatively consist of an amino acid sequence which is at least80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to, for example, thepolypeptide sequence shown in SEQ ID NO:Y, the polypeptide sequenceencoded by the cDNA contained in a deposited clone, and/or polypeptidefragments of any of these polypeptides (e.g., those fragments describedherein).

By a nucleic acid having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of the nucleicacid is identical to the reference sequence except that the nucleotidesequence may include up to five point mutations per each 100 nucleotidesof the reference nucleotide sequence encoding the polypeptide. In otherwords, to obtain a nucleic acid having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be an entire sequence shown in TableXIV, the ORF (open reading frame), or any fragment specified asdescribed herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence of the presence invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. 6:237-245(1990)). In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, anamino acid sequences shown in Table XIV (SEQ ID NO:Y) or to the aminoacid sequence encoded by cDNA contained in a deposited clone can bedetermined conventionally using known computer programs. A preferredmethod for determing the best overall match between a query sequence (asequence of the present invention) and a subject sequence, also referredto as a global sequence alignment, can be determined using the FASTDBcomputer program based on the algorithm of Brutlag et al. (Comp. App.Biosci. 6:237-245(1990)). In a sequence alignment the query and subjectsequences are either both nucleotide sequences or both amino acidsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a FASTDB amino acid alignmentare: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group Length=0, Cutoff Score=1, Window Size=sequencelength, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or thelength of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The variants may contain alterations in the coding regions, non-codingregions, or both. Especially preferred are polynucleotide variantscontaining alterations which produce silent substitutions, additions, ordeletions, but do not alter the properties or activities of the encodedpolypeptide. Nucleotide variants produced by silent substitutions due tothe degeneracy of the genetic code are preferred. Moreover, variants inwhich 5-10, 1-5, or 1-2 amino acids are substituted, delete, or added inany combination are also preferred. Polynucleotide variants can beproduced for a variety of reasons, e.g., to optimize codon expressionfor a particular host (change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

Naturally occurring variants are called “allelic variants,” and refer toone of several alternate forms of a gene occupying a given locus on achromosome of an organism. (Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985).) These allelic variants can vary at either thepolynucleotide and/or polypeptide level and are included in the presentinvention. Alternatively, non-naturally occurring variants may beproduced by mutagenesis techniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the polypeptides of the present invention. Forinstance, one or more amino acids can be deleted from the N-terminus orC-terminus of the secreted protein without substantial loss ofbiological function. The authors of Ron et al., J. Biol. Chem.268:2984-2988 (1993), reported variant KGF proteins having heparinbinding activity even after deleting 3, 8, or 27 amino-terminal aminoacid residues. Similarly, Interferon gamma exhibited up to ten timeshigher activity after deleting 8-10 amino acid residues from the carboxyterminus of this protein. (Dobeli et al., J. Biotechnology 7:199-216(1988).)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes polypeptide variants which showsubstantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity. Forexample, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie et al., Science 247:1306-1310(1990), wherein the authors indicate that there are two main strategiesfor studying the tolerance of an amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

Besides conservative amino acid substitution, variants of the presentinvention include (i) substitutions with one or more of thenon-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code, or (ii)substitution with one or more of amino acid residues having asubstituent group, or (iii) fusion of the mature polypeptide withanother compound, such as a compound to increase the stability and/orsolubility of the polypeptide (for example, polyethylene glycol), or(iv) fusion of the polypeptide with additional amino acids, such as, forexample, an IgG Fc fusion region peptide, or leader or secretorysequence, or a sequence facilitating purification. Such variantpolypeptides are deemed to be within the scope of those skilled in theart from the teachings herein.

For example, polypeptide variants containing amino acid substitutions ofcharged amino acids with other charged or neutral amino acids mayproduce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36:838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of the present invention having anamino acid sequence which contains at least one amino acid substitution,but not more than 50 amino acid substitutions, even more preferably, notmore than 40 amino acid substitutions, still more preferably, not morethan 30 amino acid substitutions, and still even more preferably, notmore than 20 amino acid substitutions. Of course, in order ofever-increasing preference, it is highly preferable for a peptide orpolypeptide to have an amino acid sequence which comprises the aminoacid sequence of the present invention, which contains at least one, butnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.In specific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of the present invention orfragments thereof (e.g., the mature form and/or other fragmentsdescribed herein), is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150,conservative amino acid substitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is also directed to polynucleotide fragments ofthe polynucleotides of the invention.

In the present invention, a “polynucleotide fragment” refers to a shortpolynucleotide having a nucleic acid sequence which: is a portion ofthat contained in a deposited clone, or encoding the polypeptide encodedby the CDNA in a deposited clone; is a portion of that shown in SEQ IDNO:X or the complementary strand thereto, or is a portion of apolynucleotide sequence encoding the polypeptide of SEQ ID NO:Y. Thenucleotide fragments of the invention are preferably at least about 15nt, and more preferably at least about 20 nt, still more preferably atleast about 30 nt, and even more preferably, at least about 40 nt, atleast about 50 nt, at least about 75 nt, or at least about 150 nt inlength. A fragment “at least 20 nt in length,” for example, is intendedto include 20 or more contiguous bases from the cDNA sequence containedin a deposited clone or the nucleotide sequence shown in SEQ ID NO:X. Inthis context “about” includes the particularly recited value, a valuelarger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at eitherterminus or at both termini. These nucleotide fragments have uses thatinclude, but are not limited to, as diagnostic probes and primers asdiscussed herein. Of course, larger fragments (e.g., 50, 150, 500, 600,2000 nucleotides) are preferred.

Moreover, representative examples of polynucleotide fragments of theinvention, include, for example, fragments comprising, or alternativelyconsisting of, a sequence from about nucleotide number 1-50, 51-100,101-150, 151-200, 201-250, 251-300, 301-350, 351-400, 401450,451-500,501-550, 551-600, 651-700, 701-750, 751-800, 800-850, 851-900, 901-950,951-1000, 1001-1050, 1051-1100, 1101-1150, 1151-1200, 1201-1250,1251-1300, 1301-1350, 1351-1400, 1401-1450, 1451-1500, 1501-1550,1551-1600, 1601-1650, 1651-1700, 1701-1750, 1751-1800, 1801-1850,1851-1900, 1901-1950, 1951-2000, or 2001 to the end of SEQ ID NO:X, orthe complementary strand thereto, or the cDNA contained in a depositedclone. In this context “about” includes the particularly recited ranges,and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides,at either terminus or at both termini. Preferably, these fragmentsencode a polypeptide which has biological activity. More preferably,these polynucleotides can be used as probes or primers as discussedherein. Polynucleotides which hybridize to these nucleic acid moleculesunder stringent hybridization conditions or lower stringency conditionsare also encompassed by the invention, as are polypeptides encoded bythese polynucleotides.

In the present invention, a “polypeptide fragment” refers to an aminoacid sequence which is a portion of that contained in SEQ ID NO:Y orencoded by the cDNA contained in a deposited clone. Protein(polypeptide) fragments may be “free-standing,” or comprised within alarger polypeptide of which the fragment forms a part or region, mostpreferably as a single continuous region. Representative examples ofpolypeptide fragments of the invention, include, for example, fragmentscomprising, or alternatively consisting of, from about amino acid number1-20, 2140, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, or 161 tothe end of the coding region. Moreover, polypeptide fragments can beabout 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150amino acids in length. In this context “about” includes the particularlyrecited ranges or values, and ranges or values larger or smaller byseveral (5, 4, 3, 2, or 1) amino acids, at either extreme or at bothextremes. Polynucleotides encoding these polypeptides are alsoencompassed by the invention.

Preferred polypeptide fragments include the secreted protein as well asthe mature form. Further preferred polypeptide fragments include thesecreted protein or the mature form having a continuous series ofdeleted residues from the amino or the carboxy terminus, or both. Forexample, any number of amino acids, ranging from 1-60, can be deletedfrom the amino terminus of either the secreted polypeptide or the matureform. Similarly, any number of amino acids, ranging from 1-30, can bedeleted from the carboxy terminus of the secreted protein or matureform. Furthermore, any combination of the above amino and carboxyterminus deletions are preferred. Similarly, polynucleotides encodingthese polypeptide fragments are also preferred.

Also preferred are polypeptide and polynucleotide fragmentscharacterized by structural or functional domains, such as fragmentsthat comprise alpha-helix and alpha-helix forming regions, beta-sheetand beta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions. Polypeptide fragments of SEQ ID NO:Y falling withinconserved domains are specifically contemplated by the presentinvention. Moreover, polynucleotides encoding these domains are alsocontemplated.

Other preferred polypeptide fragments are biologically active fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the polypeptide of thepresent invention. The biological activity of the fragments may includean improved desired activity, or a decreased undesirable activity.Polynucleotides encoding these polypeptide fragments are alsoencompassed by the invention.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a functional activity. By a polypeptidedemonstrating a “functional activity” is meant, a polypeptide capable ofdisplaying one or more known functional activities associated with afull-length (complete) polypeptide of invention protein. Such functionalactivities include, but are not limited to, biological activity,antigenicity [ability to bind (or compete with a polypeptide of theinvention for binding) to an antibody to the polypeptide of theinvention], immunogenicity (ability to generate antibody which binds toa polypeptide of the invention), ability to form multimers withpolypeptides of the invention, and ability to bind to a receptor orligand for a polypeptide of the invention.

The functional activity of polypeptides of the invention, and fragments,variants derivatives, and analogs thereof, can be assayed by variousmethods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length polypeptide of the invention forbinding to an antibody of the polypeptide of the invention, variousimmunoassays known in the art can be used, including but not limited to,competitive and non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (using colloidalgold, enzyme or radioisotope labels, for example), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. In one embodiment, antibody binding is detected bydetecting a label on the primary antibody. In another embodiment, theprimary antibody is detected by detecting binding of a secondaryantibody or reagent to the primary antibody. In a further embodiment,the secondary antibody is labeled. Many means are known in the art fordetecting binding in an immunoassay and are within the scope of thepresent invention.

In another embodiment, where a ligand for a polypeptide of the inventionidentified, or the ability of a polypeptide fragment, variant orderivative of the invention to multimerize is being evaluated, bindingcan be assayed, e.g., by means well-known in the art, such as, forexample, reducing and non-reducing gel chromatography, protein affinitychromatography, and affinity blotting. See generally, Phizicky, E., etal., 1995, Microbiol. Rev. 59:94-123. In another embodiment,physiological correlates of binding of a polypeptide of the invention toits substrates (signal transduction) can be assayed.

In addition, assays described herein (see Examples) and otherwise knownin the art may routinely be applied to measure the ability ofpolypeptides of the invention and fragments, variants derivatives andanalogs thereof to elicit related biological activity related to that ofthe polypeptide of the invention (either in vitro or in vivo). Othermethods will be known to the skilled artisan and are within the scope ofthe invention.

Epitopes & Antibodies

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:Y, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in ATCC depositNo. Z or encoded by a polynucleotide that hybridizes to the complementof the sequence of SEQ ID NO:X or contained in ATCC deposit No. Z understringent hybridization conditions or lower stringency hybridizationconditions as defined supra. The present invention further encompassespolynucleotide sequences encoding an epitope of a polypeptide sequenceof the invention (such as, for example, the sequence disclosed in SEQ IDNO:X), polynucleotide sequences of the complementary strand of apolynucleotide sequence encoding an epitope of the invention, andpolynucleotide sequences which hybridize to the complementary strandunder stringent hybridization conditions or lower stringencyhybridization conditions defined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross- reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments which function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 20, at least 25, atleast 30, at least 40, at least 50, and, most preferably, between about15 to about 30 amino acids. Preferred polypeptides comprisingimmunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acidresidues in length. Additional non-exclusive preferred antigenicepitopes include the antigenic epitopes disclosed herein, as well asportions thereof. Antigenic epitopes are useful, for example, to raiseantibodies, including monoclonal antibodies, that specifically bind theepitope. Preferred antigenic epitopes include the antigenic epitopesdisclosed herein, as well as any combination of two, three, four, fiveor more of these antigenic epitopes. Antigenic epitopes can be used asthe target molecules in inumunoassays. (See, for instance, Wilson etal., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666(1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). Preferred immunogenic epitopes include theimmunogenic epitopes disclosed herein, as well as any combination oftwo, three, four, five or more of these immunogenic epitopes. Thepolypeptides comprising one or more immunogenic epitopes may bepresented for eliciting an antibody response together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse), or, if the polypeptide is of sufficient length (at least about25 amino acids), the polypeptide may be presented without a carrier.However, immunogenic epitopes comprising as few as 8 to 10 amino acidshave been shown to be sufficient to raise antibodies capable of bindingto, at the very least, linear epitopes in a denatured polypeptide (e.g.,in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as rabbits, rats and mice are immunizedwith either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg of peptide or carrier protein and Freund's adjuvant or anyother adjuvant known for stimulating an immune response. Several boosterinjections may be needed, for instance, at intervals of about two weeks,to provide a useful titer of anti-peptide antibody which can bedetected, for example, by ELISA assay using free peptide adsorbed to asolid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention comprising an immunogenic orantigenic epitope can be fused to other polypeptide sequences. Forexample, the polypeptides of the present invention may be fused with theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. Such fusion proteins may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion desulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni2+nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:X and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide encodinga polypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:Y, and/or an epitope, ofthe present invention (as determined by immunoassays well known in theart for assaying specific antibody-antigen binding). Antibodies of theinvention include, but are not limited to, polyclonal, monoclonal,multispecific, human, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. The term “antibody,” asused herein, refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that immunospecifically binds anantigen. The immunoglobulin molecules of the invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any, animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%/o, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention under stringenthybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶M, 5×10⁻⁷ M, 10⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, ¹⁰⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferrably, antibodies of the present invention bind anantigenic epitope disclosed herein, or a portion thereof. The inventionfeatures both receptor-specific antibodies and ligand-specificantibodies. The invention also features receptor-specific antibodieswhich do not prevent ligand binding but prevent receptor activation.Receptor activation (i.e., signaling) may be determined by techniquesdescribed herein or otherwise known in the art. For example, receptoractivation can be determined by detecting the phosphorylation (e.g.,tyrosine or serine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998) Pitard et al., J. Immunol. Methods205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997);Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman etal., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N— or C— terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteniumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Example 10). In anon-limiting example, mice can be immunized with a polypeptide of theinvention or a cell expressing such peptide. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinlonan et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)2 fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework regions from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:Y.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., Bio Techniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell know in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also by used toexpress foreign polypeptides as fusion proteins with glutathioneS-transterase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa califomica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non- essential region of the viral genome (e.g., regionE1 or E3) will result in a recombinant virus that is viable and capableof expressing the antibody molecule in infected hosts. (e.g., see Logan& Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in tumcan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol.3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalently and non-covalentlyconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughliker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Feregion, or portion thereof The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fe portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fe portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP307,434; EP367,166;PCT publicationsWO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995);and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341(1992) (saidreferences incorporated by reference in their entireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:Y may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NO:Y maybe fused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature331:84-86 (1988). The polypeptides of the present invention fused orconjugated to an antibody having disulfide-linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In manycases, the Fe part in a fusion protein is beneficial in therapy anddiagnosis, and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude 125I, 131I 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, 213Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, α-interferon, β-interferon,nerve growth factor, platelet derived growth factor, tissue plasminogenactivator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See,International Publication No. WO 97/33899), AIM II (See, InternationalPublication No. WO 97/34911), Fas Ligand (Takahashi et al., Int.Immunol., 6:1567-1574 (1994)), VEGI (See, International Publication No.WO 99/23105), a thrombotic agent or an anti-angiogenic agent, e.g.,angiostatin or endostatin; or, biological response modifiers such as,for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays For Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused include but are not limited to competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP40 or Triton X-100, 1%sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate atpH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., 32P or 125I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., 3H or 125I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., 3H or 125I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein. The treatment and/or prevention of diseases,disorders, or conditions associated with aberrant expression and/oractivity of a polypeptide of the invention includes, but is not limitedto, alleviating symptoms associated with those diseases, disorders orconditions. Antibodies of the invention may be provided inpharmaceutically acceptable compositions as known in the art or asdescribed herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents).Generally, administration of products of a species origin or speciesreactivity (in the case of antibodies) that is the same species as thatof the patient is preferred. Thus, in a preferred embodiment, humanantibodies, fragments derivatives, analogs, or nucleic acids, areadministered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10⁻¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred aspect, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue- specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid- carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO93/20221). Alternatively, the nucleic acid canbe introduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contains nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdrl gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicaitonWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92 m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such asTlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription. Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic/Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred aspect, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J.Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox- like peptide which isknown to enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad.Sci. USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases, disorders,and/or conditions associated with the aberrant expression and/oractivity of a polypeptide of the invention. The invention provides forthe detection of aberrant expression of a polypeptide of interest,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell . Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

One aspect of the invention is the detection and diagnosis of a diseaseor disorder associated with aberrant expression of a polypeptide ofinterest in an animal, preferably a mammal and most preferably a human.In one embodiment, diagnosis comprises: a) administering (for example,parenterally, subcutaneously, or intraperitoneally) to a subject aneffective amount of a labeled molecule which specifically binds to thepolypeptide of interest; b) waiting for a time interval following theadministering for permitting the labeled molecule to preferentiallyconcentrate at sites in the subject where the polypeptide is expressed(and for unbound labeled molecule to be cleared to background level); c)determining background level; and d) detecting the labeled molecule inthe subject, such that detection of labeled molecule above thebackground level indicates that the subject has a particular disease ordisorder associated with aberrant expression of the polypeptide ofinterest. Background level can be determined by various methodsincluding, comparing the amount of labeled molecule detected to astandard value previously determined for a particular system.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of 99 mTc. The labeled antibody orantibody fragment will then preferentially accumulate at the location ofcells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention contain ameans for detecting the binding of an antibody to a polypeptide ofinterest (e.g., the antibody may be conjugated to a detectable substratesuch as a fluorescent compound, an enzymatic substrate, a radioactivecompound or a luminescent compound, or a second antibody whichrecognizes the first antibody may be conjugated to a detectablesubstrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface- bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

Fusion Proteins

Any polypeptide of the present invention can be used to generate fusionproteins. For example, the polypeptide of the present invention, whenfused to a second protein, can be used as an antigenic tag. Antibodiesraised against the polypeptide of the present invention can be used toindirectly detect the second protein by binding to the polypeptide.Moreover, because secreted proteins target cellular locations based ontrafficking signals, the polypeptides of the present invention can beused as targeting molecules once fused to other proteins.

Examples of domains that can be fused to polypeptides of the presentinvention include not only heterologous signal sequences, but also otherheterologous functional regions. The fusion does not necessarily need tobe direct, but may occur through linker sequences.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the polypeptide of the present invention. Forinstance, a region of additional amino acids, particularly charged aminoacids, may be added to the N-terminus of the polypeptide to improvestability and persistence during purification from the host cell orsubsequent handling and storage. Also, peptide moieties may be added tothe polypeptide to facilitate purification. Such regions may be removedprior to final preparation of the polypeptide. The addition of peptidemoieties to facilitate handling of polypeptides are familiar and routinetechniques in the art.

Moreover, polypeptides of the present invention, including fragments,and specifically epitopes, can be combined with parts of the constantdomain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1,CH2, CH3, and any combination thereof, including both entire domains andportions thereof), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. One reported example describes chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EP A 394,827; Traunecker et al., Nature 331:84-86(1988).) Fusion proteins having disulfide-linked dimeric structures (dueto the IgG) can also be more efficient in binding and neutralizing othermolecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995).)

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fe portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the polypeptides of the present invention can be fused tomarker sequences, such as a peptide which facilitates purification ofthe fused polypeptide. In preferred embodiments, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311),among others, many of which are commercially available. As described inGentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. Another peptide tag useful for purification, the “HA”tag, corresponds to an epitope derived from the influenza hemagglutininprotein. (Wilson et al., Cell 37:767 (1984).)

Thus, any of these above fusions can be engineered using thepolynucleotides or the polypeptides of the present invention.

Vectors, Host Cells, and Protein Production

The present invention also relates to vectors containing thepolynucleotide of the present invention, host cells, and the productionof polypeptides by recombinant techniques. The vector may be, forexample, a phage, plasmid, viral, or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The polynucleotide insert should be operatively linked to an appropriatepromoter, such as the phage lambda PL promoter, the E. coli lac, tip,phoA and tac promoters, the SV40 early and late promoters and promotersof retroviral LTRs, to name a few. Other suitable promoters will beknown to the skilled artisan. The expression constructs will furthercontain sites for transcription initiation, termination, and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the transcripts expressed by the constructs will preferablyinclude a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293, and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc.; pBluescript vectors, Phagescriptvectors, pNH8A, pNH16a, pNH18A, pNR46A, available from StratageneCloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia Biotech, Inc. Among preferred eukaryoticvectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available fromStratagene; and pSVK3, pBPV, pMSG and pSVL available from Phannacia.Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that the polypeptides ofthe present invention may in fact be expressed by a host cell lacking arecombinant vector.

A polypeptide of this invention can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.

Polypeptides of the present invention, and preferably the secreted form,can also be recovered from: products purified from natural sources,including bodily fluids, tissues and cells, whether directly isolated orcultured; products of chemical synthetic procedures; and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect,and mammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes. Thus, it is wellknown in the art that the N-terminal methionine encoded by thetranslation initiation codon generally is removed with high efficiencyfrom any protein after translation in all eukaryotic cells. While theN-terminal methionine on most proteins also is efficiently removed inmost prokaryotes, for some proteins, this prokaryotic removal process isinefficient, depending on the nature of the amino acid to which theN-terminal methionine is covalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., coding sequence), and/or to include geneticmaterial (e.g., heterologous polynucleotide sequences) that is operablyassociated with the polynucleotides of the invention, and whichactivates, alters, and/or amplifies endogenous polynucleotides. Forexample, techniques known in the art may be used to operably associateheterologous control regions (e.g., promoter and/or enhancer) andendogenous polynucleotide sequences via homologous recombination (see,e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; InternationalPublication No. WO 96/29411, published Sep. 26, 1996; InternationalPublication No. WO 94/12650, published Aug. 4, 1994; Koller et al.,Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); and Zijlstra et al.,Nature 342:435-438 (1989), the disclosures of each of which areincorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y., andHunkapiller et al., Nature, 310:105-111 (1984)). For example, apolypeptide corresponding to a fragment of a polypeptide sequence of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into thepolypeptide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses polypeptides which are differentially modifiedduring or after translation, e.g., by glycosylation, acetylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to an antibody molecule or othercellular ligand, etc. Any of numerous chemical modifications may becarried out by known techniques, including but not limited, to specificchemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8protease, NaBH₄; acetylation, formylation, oxidation, reduction;metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofthe polypeptides of the invention which may provide additionaladvantages such as increased solubility, stability and circulating timeof the polypeptide, or decreased immunogenicity (see U.S. Pat. No.4,179,337). The chemical moieties for derivitization may be selectedfrom water soluble polymers such as polyethylene glycol, ethyleneglycol/propylene glycol copolymers, carboxymnethylcellulose, dextran,polyvinyl alcohol and the like. The polypeptides may be modified atrandom positions within the molecule, or at predetermined positionswithin the molecule and may include one, two, three or more attachedchemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (polypeptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

The polypeptides of the invention may be in monomers or multimers (i.e.,dimers, trimers, tetramers and higher multimers). Accordingly, thepresent invention relates to monomers and multimers of the polypeptidesof the invention, their preparation, and compositions (preferably,Therapeutics) containing them. In specific embodiments, the polypeptidesof the invention are monomers, dimers, trimers or tetramers. Inadditional embodiments, the multimers of the invention are at leastdimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlypolypeptides corresponding to the amino acid sequence of SEQ ID NO:Y orencoded by the cDNA contained in a deposited clone (including fragments,variants, splice variants, and fusion proteins, corresponding to thesepolypeptides as described herein). These homomers may containpolypeptides having identical or different amino acid sequences. In aspecific embodiment, a homomer of the invention is a multimer containingonly polypeptides having an identical amino acid sequence. In anotherspecific embodiment, a homomer of the invention is a multimer containingpolypeptides having different amino acid sequences. In specificembodiments, the multimer of the invention is a homodimer (e.g.,containing polypeptides having identical or different amino acidsequences) or a homotrimer (e.g., containing polypeptides havingidentical and/or different amino acid sequences). In additionalembodiments, the homomeric multimer of the invention is at least ahomodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, theheteromeric multimer of the invention is at least a heterodimer, atleast a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the polypeptides of the invention. Suchcovalent associations may involve one or more amino acid residuescontained in the polypeptide sequence (e.g., that recited in thesequence listing, or contained in the polypeptide encoded by a depositedclone). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a fusion protein of theinvention.

In one example, covalent associations are between the heterologoussequence contained in a fusion protein of the invention (see, e.g., U.S.Pat. No. 5,478,925). In a specific example, the covalent associationsare between the heterologous sequence contained in an Fc fusion proteinof the invention (as described herein). In another specific example,covalent associations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another protein that is capableof forming covalently associated multimers, such as for example,oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305,the contents of which are herein incorporated by reference in itsentirety). In another embodiment, two or more polypeptides of theinvention are joined through peptide linkers. Examples include thosepeptide linkers described in U.S. Pat. No. 5,073,627 (herebyincorporated by reference). Proteins comprising multiple polypeptides ofthe invention separated by peptide linkers may be produced usingconventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hyrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Uses of the Polynucleotides

Each of the polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

The polynucleotides of the present invention are useful for chromosomeidentification. There exists an ongoing need to identify new chromosomemarkers, since few chromosome marking reagents, based on actual sequencedata (repeat polymnorphisms), are presently available. Eachpolynucleotide of the present invention can be used as a chromosomemarker.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:X. Primerscan be selected using computer analysis so that primers do not span morethan one predicted exon in the genomic DNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the SEQ ID NO:X will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the polynucleotides can be achieved with panels ofspecific chromosome fragments. Other gene mapping strategies that can beused include in situ hybridization, prescreening with labeledflow-sorted chromosomes, and preselection by hybridization to constructchromosome specific-cDNA libraries.

Precise chromosomal location of the polynucleotides can also be achievedusing fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Venma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the polynucleotides can be used individually (tomark a single chromosome or a single site on that chromosome) or inpanels (for marking multiple sites and/or multiple chromosomes).Preferred polynucleotides correspond to the noncoding regions of thecDNAs because the coding sequences are more likely conserved within genefamilies, thus increasing the chance of cross hybridization duringchromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library) .) Assuming 1 megabase mapping resolution and onegene per 20 kb, a CDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

Thus, once coinheritance is established, differences in thepolynucleotide and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the polypeptide and the corresponding gene fromseveral normal individuals is required to distinguish the mutation froma polymorphism. If a new polymorphism is identified, this polymorphicpolypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedindividuals as compared to unaffected individuals can be assessed usingpolynucleotides of the present invention. Any of these alterations(altered expression, chromosomal rearrangement, or mutation) can be usedas a diagnostic or prognostic marker.

Thus, the invention also provides a diagnostic method useful duringdiagnosis of a disorder, involving measuring the expression level ofpolynucleotides of the present invention in cells or body fluid from anindividual and comparing the measured gene expression level with astandard level of polynucleotide expression level, whereby an increaseor decrease in the gene expression level compared to the standard isindicative of a disorder.

In still another embodiment, the invention includes a kit for analyzingsamples for the presence of proliferative and/or cancerouspolynucleotides derived from a test subject. In a general embodiment,the kit includes at least one polynucleotide probe containing anucleotide sequence that will specifically hybridize with apolynucleotide of the present invention and a suitable container. In aspecific embodiment, the kit includes two polynucleotide probes definingan internal region of the polynucleotide of the present invention, whereeach probe has one strand containing a 31′ mer-end internal to theregion. In a further embodiment, the probes may be useful as primers forpolymerase chain reaction amplification.

Where a diagnosis of a disorder, has already been made according toconventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting enhanced or depressedpolynucleotide of the present invention expression will experience aworse clinical outcome relative to patients expressing the gene at alevel nearer the standard level.

By “measuring the expression level of polynucleotide of the presentinvention” is intended qualitatively or quantitatively measuring orestimating the level of the polypeptide of the present invention or thelevel of the mRNA encoding the polypeptide in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or MRNA level) or relatively (e.g., by comparing to thepolypeptide level or mRNA level in a second biological sample).Preferably, the polypeptide level or MRNA level in the first biologicalsample is measured or estimated and compared to a standard polypeptidelevel or MRNA level, the standard being taken from a second biologicalsample obtained from an individual not having the disorder or beingdetermined by averaging levels from a population of individuals nothaving a disorder. As will be appreciated in the art, once a standardpolypeptide level or mRNA level is known, it can be used repeatedly as astandard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains the polypeptide of the present invention or mRNA. Asindicated, biological samples include body fluids (such as semen, lymph,sera, plasma, urine, synovial fluid and spinal fluid) which contain thepolypeptide of the present invention, and other tissue sources found toexpress the polypeptide of the present invention. Methods for obtainingtissue biopsies and body fluids from mammals are well known in the art.Where the biological sample is to include mRNA, a tissue biopsy is thepreferred source.

The method(s) provided above may preferably be applied in a diagnosticmethod and/or kits in which polynucleotides and/or polypeptides areattached to a solid support. In one exemplary method, the support may bea “gene chip” or a “biological chip” as described in U.S. Pat. Nos.5,837,832, 5,874,219, and 5,856,174. Further, such a gene chip withpolynucleotides of the present invention attached may be used toidentify polymorphisms between the polynucleotide sequences, withpolynucleotides isolated from a test subject. The knowledge of suchpolymorphisms (i.e. their location, as well as, their existence) wouldbe beneficial in identifying disease loci for many disorders, includingcancerous diseases and conditions. Such a method is described in U.S.Pat. Nos. 5,858,659 and 5,856,104. The US Patents referenced supra arehereby incorporated by reference in their entirety herein.

The present invention encompasses polynucleotides of the presentinvention that are chemically synthesized, or reproduced as peptidenucleic acids (PNA), or according to other methods known in the art. Theuse of PNAs would serve as the preferred form if the polynucleotides areincorporated onto a solid support, or gene chip. For the purposes of thepresent invention, a peptide nucleic acid (PNA) is a polyamide type ofDNA analog and the monomeric units for adenine, guanine, thymine andcytosine are available commercially (Perceptive Biosystems). Certaincomponents of DNA, such as phosphorus, phosphorus oxides, or deoxyribosederivatives, are not present in PNAs. As disclosed by P. E. Nielsen, M.Egholm, R. H. Berg and O. Buchardt, Science 254, 1497 (1991); and M.Egholm, O. Buchardt, L. Christensen, C. Behrens, S. M. Freier, D. A.Driver, R. H. Berg, S. K. Kim, B. Norden, and P. E. Nielsen, Nature 365,666 (1993), PNAs bind specifically and tightly to complementary DNAstrands and are not degraded by nucleases. In fact, PNA binds morestrongly to DNA than DNA itself does. This is probably because there isno electrostatic repulsion between the two strands, and also thepolyamide backbone is more flexible. Because of this, PNA/DNA duplexesbind under a wider range of stringency conditions than DNA/DNA duplexes,making it easier to perform multiplex hybridization. Smaller probes canbe used than with DNA due to the strong binding. In addition, it is morelikely that single base mismatches can be determined with PNA/DNAhybfidization because a single mismatch in a PNA/DNA 15-mer lowers themelting point (T.sub.m) by 8°-20° C., vs. 4°-16° C. for the DNA/DNA15-mer duplex. Also, the absence of charge groups in PNA means thathybridization can be done at low ionic strengths and reduce possibleinterference by salt during the analysis.

The present invention is useful for detecting cancer in mammals. Inparticular the invention is useful during diagnosis of pathological cellproliferative neoplasias which include, but are not limited to: acutemyelogenous leukemias including acute monocytic leukemia, acutemyeloblastic leukemia, acute promyelocytic leukemia, acutemyelomonocytic leukemia, acute erythroleukemia, acute megakaryocyticleukemia, and acute undifferentiated leukemia, etc.; and chronicmyelogenous leukemias including chronic myelomonocytic leukemia, chronicgranulocytic leukemia, etc. Preferred mammals include monkeys, apes,cats, dogs, cows, pigs, horses, rabbits and humans. Particularlypreferred are humans.

Pathological cell proliferative disorders are often associated withinappropriate activation of proto-oncogenes. (Gelmann, E. P. et al.,“The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,”in Neoplastic Diseases of the Blood, Vol 1., Wiemiik, P. H. et al. eds.,161-182 (1985)). Neoplasias are now believed to result from thequalitative alteration of a normal cellular gene product, or from thequantitative modification of gene expression by insertion into thechromosome of a viral sequence, by chromosomal translocation of a geneto a more actively transcribed region, or by some other mechanism.(Gelmann et al., supra) It is likely that mutated or altered expressionof specific genes is involved in the pathogenesis of some leukemias,among other tissues and cell types. (Gelmann et al., supra) Indeed, thehuman counterparts of the oncogenes involved in some animal neoplasiashave been amplified or translocated in some cases of human leukemia andcarcinoma. (Gelmann et al., supra)

For example, c-myc expression is highly amplified in the non-lymphocyticleukemia cell line HL-60. When HL-60 cells are chemically induced tostop proliferation, the level of c-myc is found to be downregulated.(International Publication Number WO 91/15580) However, it has beenshown that exposure of HL-60 cells to a DNA construct that iscomplementary to the 5′ end of c-myc or c-myb blocks translation of thecorresponding mRNAs which downregulates expression of the c-myc or c-mybproteins and causes arrest of cell proliferation and differentiation ofthe treated cells. (International Publication Number WO 91/15580;Wickstrom et al., Proc. Natl. Acad. Sci. 85:1028 (1988); Anfossi et al.,Proc. Natl. Acad. Sci. 86:3379 (1989)). However, the skilled artisanwould appreciate the present invention's usefulness would not be limitedto treatment of proliferative disorders of hematopoietic cells andtissues, in light of the numerous cells and cell types of varyingorigins which are known to exhibit proliferative phenotypes.

In addition to the foregoing, a polynucleotide can be used to controlgene expression through triple helix formation or antisense DNA or RNA.Antisense techniques are discussed, for example, in Okano, J. Neurochem.56:560 (1991); “Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRCPress, Boca Raton, Fla. (1988). Triple helix formation isdiscussed in, for instance Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241: 456 (1988); and Dervan et al.,Science 251:1360 (1991). Both methods rely on binding of thepolynucleotide to a complementary DNA or RNA. For these techniques,preferred polynucleotides are usually oligonucleotides 20 to 40 bases inlength and complementary to either the region of the gene involved intranscription (triple helix—see Lee et al., Nucl. Acids Res. 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991) ) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat disease.

Polynucleotides of the present invention are also useful in genetherapy. One goal of gene therapy is to insert a normal gene into anorganism having a defective gene, in an effort to correct the geneticdefect. The polynucleotides disclosed in the present invention offer ameans of targeting such genetic defects in a highly accurate manner.Another goal is to insert a new gene that was not present in the hostgenome, thereby producing a new trait in the host cell.

The polynucleotides are also useful for identifying individuals fromminute biological samples. The United States military, for example, isconsidering the use of restriction fragment length polymorphism (RFLP)for identification of its personnel. In this technique, an individual'sgenomic DNA is digested with one or more restriction enzymes, and probedon a Southern blot to yield unique bands for identifying personnel. Thismethod does not suffer from the current limitations of “Dog Tags” whichcan be lost, switched, or stolen, making positive identificationdifficult. The polynucleotides of the present invention can be used asadditional DNA markers for RFLP.

The polynucleotides of the present invention can also be used as analternative to RFLP, by determining the actual base-by-base DNA sequenceof selected portions of an individual's genome. These sequences can beused to prepare PCR primers for amplifying and isolating such selectedDNA, which can then be sequenced. Using this technique, individuals canbe identified because each individual will have a unique set of DNAsequences. Once an unique ED database is established for an individual,positive identification of that individual, living or dead, can be madefrom extremely small tissue samples.

Forensic biology also benefits from using DNA-based identificationtechniques as disclosed herein. DNA sequences taken from very smallbiological samples such as tissues, e.g., hair or skin, or body fluids,e.g., blood, saliva, semen, synovial fluid, amniotic fluid, breast milk,lymph, pulmonary sputum or surfactant,urine,fecal matter, etc., can beamplified using PCR. In one prior art technique, gene sequencesamplified from polymorphic loci, such as DQa class 11 HLA gene, are usedin forensic biology to identify individuals. (Erlich, H., PCRTechnology, Freeman and Co. (1992).) Once these specific polymorphicloci are amplified, they are digested with one or more restrictionenzymes, yielding an identifying set of bands on a Southern blot probedwith DNA corresponding to the DQa class II HLA gene. Similarly,polynucleotides of the present invention can be used as polymorphicmarkers for forensic purposes.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, in forensics whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, for example, DNA probes or primers specific to particulartissue prepared from the sequences of the present invention. Panels ofsuch reagents can identify tissue by species and/or by organ type. In asimilar fashion, these reagents can be used to screen tissue culturesfor contamination.

In the very least, the polynucleotides of the present invention can beused as molecular weight markers on Southern gels, as diagnostic probesfor the presence of a specific MRNA in a particular cell type, as aprobe to “subtract-out” known sequences in the process of discoveringnovel polynucleotides, for selecting and making oligomers for attachmentto a “gene chip” or other support, to raise anti-DNA antibodies usingDNA immunization techniques, and as an antigen to elicit an immuneresponse.

Uses of the Polypeptides

Each of the polypeptides identified herein can be used in numerous ways.The following description should be considered exemplary and utilizesknown techniques.

A polypeptide of the present invention can be used to assay proteinlevels in a biological sample using antibody-based techniques. Forexample, protein expression in tissues can be studied with classicalimmunohistological methods. (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell . Biol. 105:3087-3096(1987).) Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase, and radioisotopes, such as iodine (125I, 121I), carbon (14C),sulfur (35S), tritium (3H), indium (112In), and technetium (99 mTc), andfluorescent labels, such as fluorescein and rhodamine, and biotin.

In addition to assaying secreted protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99 mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99 mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of a polypeptide of the presentinvention in cells or body fluid of an individual; (b) comparing thelevel of gene expression with a standard gene expression level, wherebyan increase or decrease in the assayed polypeptide gene expression levelcompared to the standard expression level is indicative of a disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Moreover, polypeptides of the present invention can be used to treatdisease. For example, patients can be administered a polypeptide of thepresent invention in an effort to replace absent or decreased levels ofthe polypeptide (e.g., insulin), to supplement absent or decreasedlevels of a different polypeptide (e.g., hemoglobin S for hemoglobin B,SOD, catalase, DNA repair proteins), to inhibit the activity of apolypeptide (e.g., an oncogene or tumor supressor), to activate theactivity of a polypeptide (e.g., by binding to a receptor), to reducethe activity of a membrane bound receptor by competing with it for freeligand (e.g., soluble TNF receptors used in reducing inflammation), orto bring about a desired response (e.g., blood vessel growth inhibition,enhancement of the immune response to proliferative cells or tissues).

Similarly, antibodies directed to a polypeptide of the present inventioncan also be used to treat disease. For example, administration of anantibody directed to a polypeptide of the present invention can bind andreduce overproduction of the polypeptide. Similarly, administration ofan antibody can activate the polypeptide, such as by binding to apolypeptide bound to a membrane (receptor).

At the very least, the polypeptides of the present invention can be usedas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods well known to those of skill in theart. Polypeptides can also be used to raise antibodies, which in turnare used to measure protein expression from a recombinant cell, as a wayof assessing transformation of the host cell. Moreover, the polypeptidesof the present invention can be used to test the following biologicalactivities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating disorders, diseases and conditions. The gene therapy methodsrelate to the introduction of nucleic acid (DNA, RNA and antisense DNAor RNA) sequences into an animal to achieve expression of a polypeptideof the present invention. This method requires a polynucleotide whichcodes for a polypeptide of the invention that operatively linked to apromoter and any other genetic elements necessary for the expression ofthe polypeptide by the target tissue. Such gene therapy and deliverytechniques are known in the art, see, for example, WO90/11092, which isherein incorporated by reference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to apolynucleotide of the invention ex vivo, with the engineered cells thenbeing provided to a patient to be treated with the polypeptide. Suchmethods are well-known in the art. For example, see Belldegrun et al.,J. Natl. Cancer Inst., 85:207-216 (1993); Ferrantini et al., CancerResearch, 53:107-1112 (1993); Ferrantini et al., J. Immunology153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229(1995); Ogura et al., Cancer Research 50:5102-5106 (1990); Santodonato,et al., Human Gene Therapy 7:1-10 (1996); Santodonato, et al., GeneTherapy 4:1246-1255 (1997); and Zhang, et al., Cancer Gene Therapy3:31-38 (1996)), which are herein incorporated by reference. In oneembodiment, the cells which are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

As discussed in more detail below, the polynucleotide constructs can bedelivered by any method that delivers injectable materials to the cellsof an animal, such as, injection into the interstitial space of tissues(heart, muscle, skin, lung, liver, and the like). The polynucleotideconstructs may be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

In one embodiment, the polynucleotide of the invention is delivered as anaked polynucleotide. The term “naked” polynucleotide, DNA or RNA refersto sequences that are free from any delivery vehicle that acts toassist, promote or facilitate entry into the cell, including viralsequences, viral particles, liposome formulations, lipofectin orprecipitating agents and the like. However, the polynucleotides of theinvention can also be delivered in liposome formulations and lipofectinformulations and the like can be prepared by methods well known to thoseskilled in the art. Such methods are described, for example, in U.S.Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are hereinincorporated by reference.

The polynucleotide vector constructs of the invention used in the genetherapy method are preferably constructs that will not integrate intothe host genome nor will they contain sequences that allow forreplication. Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL availablefrom Pharmacia; and pEF1/V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of polynucleotide sequence of the invention.Suitable promoters include adenoviral promoters, such as the adenoviralmajor late promoter; or heterologous promoters, such as thecytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV)promoter; inducible promoters, such as the MMT promoter, themetallothionein promoter; heat shock promoters; the albumin promoter;the ApoAI promoter; human globin promoters; viral thymidine kinasepromoters, such as the Herpes Simplex thymidine kinase promoter;retroviral LTRs; the b-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter for thepolynucleotides of the invention.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The polynucleotide construct of the invention can be delivered to theinterstitial space of tissues within the an animal, including of muscle,skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph,blood, bone, cartilage, pancreas, kidney, gall bladder, stomach,intestine, testis, ovary, uterus, rectum, nervous system, eye, gland,and connective tissue. Interstitial space of the tissues comprises theintercellular, fluid, mucopolysaccharide matrix among the reticularfibers of organ tissues, elastic fibers in the walls of vessels orchambers, collagen fibers of fibrous tissues, or that same matrix withinconnective tissue ensheathing muscle cells or in the lacunae of bone. Itis similarly the space occupied by the plasma of the circulation and thelymph fluid of the lymphatic channels. Delivery to the interstitialspace of muscle tissue is preferred for the reasons discussed below.They may be conveniently delivered by injection into the tissuescomprising these cells. They are preferably delivered to and expressedin persistent, non-dividing cells which are differentiated, althoughdelivery and expression may be achieved in non-differentiated or lesscompletely differentiated cells, such as, for example, stem cells ofblood or skin fibroblasts. In vivo muscle cells are particularlycompetent in their ability to take up and express polynucleotides.

For the nakednucleic acid sequence injection, an effective dosage amountof DNA or RNA will be in the range of from about 0.05 mg/kg body weightto about 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the arl,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the polynucleotide constructs of the inventionare complexed in a liposome preparation. Liposomal preparations for usein the instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA ,84:7413-7416 (1987), which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA, 86:6077-6081 (1989), whichis herein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem., 265:10189-10192 (1990), which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl Acad. Sci. USA , 84:7413-7416 (1987), which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication NO: WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., Felgner etal., Proc. Natl. Acad. Sci. USA, 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology , 101:512-527 (1983), which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta, 394:483 (1975); Wilsonet al., Cell , 17:77 (1979)); ether injection (Deamer et al., Biochim.Biophys. Acta, 443:629 (1976); Ostro et al., Biochem. Biophys. Res.Commun., 76:836 (1977); Fraley et al., Proc. Natl. Acad. Sci. USA,76:3348 (1979)); detergent dialysis (Enoch et al., Proc. Natl. Acad.Sci. USA , 76:145 (1979)); and reverse-phase evaporation (REV) (Fraleyet al., J. Biol. Chem., 255:10431 (1980); Szoka et al., Proc. Natl.Acad. Sci. USA, 75:145 (1978); Schaefer-Ridder et al., Science, 215:166(1982)), which are herein incorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication NO: WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication NO: WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are engineered, ex vivo or in vivo, usinga retroviral particle containing RNA which comprises a sequence encodingpolypeptides of the invention. Retroviruses from which the retroviralplasmid vectors may be derived include, but are not limited to, MoloneyMurine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, HarveySarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, humanimmunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammarytumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy, 1:5-14(1990), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding polypeptides of the invention.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express polypeptides of the invention.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with polynucleotides of the invention contained in an adenovirus vector.Adenovirus can be manipulated such that it encodes and expressespolypeptides of the invention, and at the same time is inactivated interms of its ability to replicate in a normal lytic viral life cycle.Adenovirus expression is achieved without integration of the viral DNAinto the host cell chromosome, thereby alleviating concerns aboutinsertional mutagenesis. Furthermore, adenoviruses have been used aslive enteric vaccines for many years with an excellent safety profile(Schwartzet al., Am. Rev. Respir. Dis., 109:233-238 (1974)). Finally,adenovirus mediated gene transfer has been demonstrated in a number ofinstances including transfer of alpha-1-antitrypsin and CFTR to thelungs of cotton rats (Rosenfeld et al., Science, 252:431-434 (1991);Rosenfeld et al., Cell, 68:143-155 (1992)). Furthermore, extensivestudies to attempt to establish adenovirus as a causative agent in humancancer were uniformly negative (Green et al. Proc. Natl. Acad. Sci. USA, 76:6606 (1979)).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel., 3:499-503 (1993); Rosenfeld etal., Cell , 68:143-155 (1992);Engelhardt etal., Human Genet. Ther., 4:759-769 (1993); Yang et al.,Nature Genet., 7:362-369 (1994); Wilson et al., Nature, 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express Ela and Elb, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, but cannot replicate in most cells. Replication deficientadenoviruses may be deleted in one or more of all or a portion of thefollowing genes: E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, Curr. Topics in Microbiol. Immunol.,158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The polynucleotide constructcontaining polynucleotides of the invention is inserted into the AAVvector using standard cloning methods, such as those found in Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press(1989). The recombinant AAV vector is then transfected into packagingcells which are infected with a helper virus, using any standardtechnique, including lipofection, electroporation calcium phosphateprecipitation, etc. Appropriate helper viruses include adenoviruses,cytomegaloviruses, vaccinia viruses, or herpes viruses. Once thepackaging cells are transfected and infected, they will produceinfectious AAV viral particles which contain the polynucleotideconstruct of the invention. These viral particles are then used totransduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the polynucleotide construct integrated into itsgenome, and will express the desired gene product.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding the polypeptide sequence of interest) via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication NO: WO 96/29411, published Sep. 26, 1996;International Publication NO: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot normally expressed in the cells, or is expressed at a lower levelthan desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the desired endogenous polynucleotide sequence so thepromoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous sequence is placed underthe control of the promoter. The promoter then drives the expression ofthe endogenous sequence.

The polynucleotides encoding polypeptides of the present invention maybe administered along with other polynucleotides encoding otherangiongenic proteins. Angiogenic proteins include, but are not limitedto, acidic and basic fibroblast growth factors, VEGF-1, VEGF-2 (VEGF-C),VEGF-3 (VEGF-B), epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

Preferably, the polynucleotide encoding a polypeptide of the inventioncontains a secretory signal sequence that facilitates secretion of theprotein. Typically, the signal sequence is positioned in the codingregion of the polynucleotide to be expressed towards or at the 5′ end ofthe coding region. The signal sequence may be homologous or heterologousto the polynucleotide of interest and may be homologous or heterologousto the cells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers. (Kaneda et al.,Science, 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA,189:11277-11281 (1992), which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian. Therapeuticcompositions of the present invention can be administered to any animal,preferably to mammals and birds. Preferred mammals include humans, dogs,cats, mice, rats, rabbits sheep, cattle, horses and pigs, with humansbeing particularly

Biological Activities

The polynucleotides or polypeptides, or agonists or antagonists of thepresent invention can be used in assays to test for one or morebiological activities. If these polynucleotides and polypeptides doexhibit activity in a particular assay, it is likely that thesemolecules may be involved in the diseases associated with the biologicalactivity. Thus, the polynucleotides or polypeptides, or agonists orantagonists could be used to treat the associated disease.

Immune Activity

Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, and/or diagnosing diseases, disorders, and/or conditions ofthe immune system, by, for example, activating or inhibiting theproliferation, differentiation, or mobilization (chemotaxis) of immunecells. Immune cells develop through a process called hematopoiesis,producing myeloid (platelets, red blood cells, neutrophils, andmacrophages) and lymphoid (B and T lymphocytes) cells from pluripotentstem cells. The etiology of these immune diseases, disorders, and/orconditions may be genetic, somatic, such as cancer and some autoimmunediseases, acquired (e.g., by chemotherapy or toxins), or infectious.Moreover, polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention can be used as a marker or detectorof a particular immune system disease or disorder.

Polynuclebtides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, and/or diagnosing diseases, disorders, and/or conditions ofhematopoietic cells. Polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention could be used toincrease differentiation and proliferation of hematopoietic cells,including the pluripotent stem cells, in an effort to treat or preventthose diseases, disorders, and/or conditions associated with a decreasein certain (or many) types hematopoietic cells. Examples of immunologicdeficiency syndromes include, but are not limited to: blood proteindiseases, disorders, and/or conditions (e.g., agammaglobulinemia,dysgammaglobulinemia), ataxia telangiectasia, common variableimmunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection,leukocyte adhesion deficiency syndrome, lymphopenia, phagocytebactericidal dysfunction, severe combined immunodeficiency (SCIDs),Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.

Moreover, polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention could also be used to modulatehemostatic (the stopping of bleeding) or thrombolytic activity (clotformation). For example, by increasing hemostatic or thrombolyticactivity, polynucleotides or polypeptides, and/or agonists orantagonists of the present invention could be used to treat or preventblood coagulation diseases, disorders, and/or conditions (e.g.,afibrinogenemia, factor deficiencies), blood platelet diseases,disorders, and/or conditions (e.g., thrombocytopenia), or woundsresulting from trauma, surgery, or other causes. Alternatively,polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention that can decrease hemostatic orthrombolytic activity could be used to inhibit or dissolve clotting.These molecules could be important in the treatment or prevention ofheart attacks (infarction), strokes, or scarring.

The polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be useful in treating,preventing, and/or diagnosing autoimmune disorders. Many autoimmunedisorders result from inappropriate recognition of self as foreignmaterial by immune cells. This inappropriate recognition results in animmune response leading to the destruction of the host tissue.Therefore, the administration of polynucleotides and polypeptides of theinvention that can inhibit an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

Autoimmune diseases or disorders that may be treated, prevented, and/ordiagnosed by polynucleotides, polypeptides, antibodies, and/or agonistsor antagonists of the present invention include, but are not limited to,one or more of the following: autoimmune hemolytic anemia, autoimmuneneonatal thrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,demmatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgAnephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation,Autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitis,and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism(i.e., Hashimoto's thyroiditis, systemic lupus erhythematosus,Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, forexample, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulinresistance, autoinunune hemolytic anemia, autoimmune thrombocytopenicpurpura, rheumatoid arthritis, schieroderma with anti-collagenantibodies, mixed connective tissue disease,polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison'sdisease, infertility, glomerulonephritis such as primaryglomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren'ssyndrome, diabetes millitus, and adrenergic drug resistance (includingadrenergic drug resistance with asthma or cystic fibrosis), chronicactive hepatitis, primary biliary cirrhosis, other endocrine glandfailure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria,atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulamatous, degenerative, and atrophic disorders.

Additional autoimmune disorders (that are probable) that may be treated,prevented, and/or diagnosed with the compositions of the inventioninclude, but are not limited to, rheumatoid arthritis (oftencharacterized, e.g., by immune complexes in joints), scleroderma withanti-collagen antibodies (often characterized, e.g., by nucleolar andother nuclear antibodies), mixed connective tissue disease (oftencharacterized, e.g., by antibodies to extractable nuclear antigens(e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., bynonhistone ANA), pernicious anemia (often characterized, e.g., byantiparietal cell, microsomes, and intrinsic factor antibodies),idiopathic Addison's disease (often characterized, e.g., by humoral andcell-mediated adrenal cytotoxicity, infertility (often characterized,e.g., by antispermatozoal antibodies), glomerulonephritis (oftencharacterized, e.g., by glomerular basement membrane antibodies orimmune complexes), bullous pemphigoid (often characterized, e.g., by IgGand complement in basement membrane), Sjogren's syndrome (oftencharacterized, e.g., by multiple tissue antibodies, and/or a specificnonhistone ANA (SS-B)), diabetes millitus (often characterized, e.g., bycell-mediated and humoral islet cell antibodies), and adrenergic drugresistance (including adrenergic drug resistance with asthma or cysticfibrosis) (often characterized, e.g., by beta-adrenergic receptorantibodies).

Additional autoimmune disorders (that are possible) that may be treated,prevented, and/or diagnosed with the compositions of the inventioninclude, but are not limited to, chronic active hepatitis (oftencharacterized, e.g., by smooth muscle antibodies), primary biliarycirrhosis (often characterized, e.g., by mitchondrial antibodies), otherendocrine gland failure (often characterized, e.g., by specific tissueantibodies in some cases), vitiligo (often characterized, e.g., bymelanocyte antibodies), vasculitis (often characterized, e.g., by Ig andcomplement in vessel walls and/or low serum complement), post-MI (oftencharacterized, e.g., by myocardial antibodies), cardiotomy syndrome(often characterized, e.g., by myocardial antibodies), urticaria (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), atopicdermatitis (often characterized, e.g., by IgG and IgM antibodies toIgE), asthma (often characterized, e.g., by IgG and IgM antibodies toIgE), and many other inflammatory, granulamatous, degenerative, andatrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, and/or diagnosed using for example, antagonists oragonists, pqlypeptides or polynucleotides, or antibodies of the presentinvention.

In a preferred embodiment polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention could be used asan agent to boost immunoresponsiveness among B cell and/or T cellimmunodeficient individuals.

B cell immunodeficiencies that may be ameliorated or treated byadministering the polypeptides or polynucleotides of the invention,and/or agonists thereof, include, but are not limited to, severecombined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosinedeaminase deficiency (ADA deficiency), X-linked agammaglobulinemia(XLA), Bruton's disease, congenital agammaglobulinemia, X-linkedinfantile agammaglobulinemia, acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, transient hypogammaglobulinemia of infancy,unspecified hypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVI) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell Iymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymophoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

T cell deficiencies that may be ameliorated or treated by administeringthe polypeptides or polynucleotides of the invention, and/or agoniststhereof include, but are not limited to, for example, DiGeorge anomaly,thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22q11.2deletion, chronic mucocutaneous candidiasis, natural killer celldeficiency (NK), idiopathic CD4+ T-lymphocytopenia, immunodeficiencywith predominant T cell defect (unspecified), and unspecifiedimmunodeficiency of cell mediated immunity. In specific embodiments,DiGeorge anomaly or conditions associated with DiGeorge anomaly areameliorated or treated by, for example, administering the polypeptidesor polynucleotides of the invention, or antagonists or agonists thereof.

Other immunodeficiencies that may be ameliorated or treated byadministering polypeptides or polynucleotides of the invention, and/oragonists thereof, include, but are not limited to, severe combinedimmunodeficiency (SCID; e.g., X-linked SCID, autosomal SCID, andadenosine deaminase deficiency), ataxia-telangiectasia, Wiskott-Aldrichsyndrome, short-limber dwarfism, X-linked lymphoproliferative syndrome(XLP), Nezelof syndrome (e.g., purine nucleoside phosphorylasedeficiency), MHC Class II deficiency. In specific embodiments,ataxia-telangiectasia or conditions associated withataxia-telangiectasia are ameliorated or treated by administering thepolypeptides or polynucleotides of the invention, and/or agoniststhereof.

In a specific preferred embodiment, rheumatoid arthritis is treated,prevented, and/or diagnosed using polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present invention. Inanother specific preferred embodiment, systemic lupus erythemosus istreated, prevented, and/or diagnosed using polynucleotides,polypeptides, antibodies, and/or agonists or antagonists of the presentinvention. In another specific preferred embodiment, idiopathicthrombocytopenia purpura is treated, prevented, and/or diagnosed usingpolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention. In another specific preferredembodiment IgA nephropathy is treated, prevented, and/or diagnosed usingpolynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention. In a preferred embodiment, theautoimmune diseases and disorders and/or conditions associated with thediseases and disorders recited above are treated, prevented, and/ordiagnosed using antibodies against the protein of the invention.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, prevented, and/or diagnosed using polypeptides, antibodies,or polynucleotides of the invention, and/or agonists or antagoniststhereof Moreover, these molecules can be used to treat, prevent, and/ordiagnose anaphylaxis, hypersensitivity to an antigenic molecule, orblood group incompatibility.

Moreover, inflammatory conditions may also be treated, diagnosed, and/orprevented with polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention. Such inflammatoryconditions include, but are not limited to, for example, respiratorydisorders (such as, e.g., asthma and allergy); gastrointestinaldisorders (such as, e.g., inflammatory bowel disease); cancers (such as,e.g., gastric, ovarian, lung, bladder, liver, and breast); CNS disorders(such as, e.g., multiple sclerosis, blood-brain barrier permeability,ischemic brain injury and/or stroke, traumatic brain injury,neurodegenerative disorders (such as, e.g., Parkinson's disease andAlzheimer's disease), AIDS-related dementia, and prion disease);cardiovascular disorders (such as, e.g., atherosclerosis, myocarditis,cardiovascular disease, and cardiopulmonary bypass complications); aswell as many additional diseases, conditions, and disorders that arecharacterized by inflammation (such as, e.g., chronic hepatitis (B andC), rheumatoid arthritis, gout, trauma, septic shock, pancreatitis,sarcoidosis, dermatitis, renal ischemia-reperfusion injury, Grave'sdisease, systemic lupus erythematosis, diabetes mellitus (i.e., type 1diabetes), and allogenic transplant rejection).

In specific embodiments, polypeptides, antibodies, or polynucleotides ofthe invention, and/or agonists or antagonists thereof, are useful totreat, diagnose, and/or prevent transplantation rejections,graft-versus-host disease, autoimmune and inflammatory diseases (e.g.,immune complex-induced vasculitis, glomerulonephritis, hemolytic anemia,myasthenia gravis, type II collagen-induced arthritis, experimentalallergic and hyperacute xenograft rejection, rheumatoid arthritis, andsystemic lupus erythematosus (SLE). Organ rejection occurs by hostimmune cell destruction of the transplanted tissue through an immuneresponse. Similarly, an immune response is also involved in GVHD, but,in this case, the foreign transplanted immune cells destroy the hosttissues. Polypeptides, antibodies, or polynucleotides of the invention,and/or agonists or antagonists thereof, that inhibit an immune response,particularly the activation, proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

Similarly, polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may also be used to modulate and/ordiagnose inflammation. For example, since polypeptides, antibodies, orpolynucleotides of the invention, and/or agonists or antagonists of theinvention may inhibit the activation, proliferation and/ordifferentiation of cells involved in an inflammatory response, thesemolecules can be used to treat, diagnose, or prognose, inflammatoryconditions, both chronic and acute conditions, including, but notlimited to, inflammation associated with infection (e.g., septic shock,sepsis, or systemic inflammatory response syndrome (SIRS)),ischemia-reperfusion injury, endotoxin lethality, arthritis,complement-mediated hyperacute rejection, nephritis, cytokine orchemokine induced lung injury, inflammatory bowel disease, Croln'sdisease, and resulting from over production of cytokines (e.g., TNF orIL-1.).

Polypeptides, antibodies, polynucleotides and/or agonists or antagonistsof the invention can be used to treat, detect, and/or prevent infectiousagents. For example, by increasing the immune response, particularlyincreasing the proliferation activation and/or differentiation of Band/or T cells, infectious diseases may be treated, detected, and/orprevented. The immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention may also directlyinhibit the infectious agent (refer to section of application listinginfectious agents, etc), without necessarily eliciting an immuneresponse.

Additional preferred embodiments of the invention include, but are notlimited to, the use of polypeptides, antibodies, polynucleotides and/oragonists or antagonists in the following applications:

Administration to an animal (e.g., mouse, rat, rabbit, hamster, guineapig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat,non-human primate, and human, most preferably human) to boost the immunesystem to produce increased quantities of one or more antibodies (e.g.,IgG, IgA, IgM, and IgE), to induce higher affinity antibody production(e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response.

Administration to an animal (including, but not limited to, those listedabove, and also including transgenic animals) incapable of producingfunctional endogenous antibody molecules or having an otherwisecompromised endogenous immune system, but which is capable of producinghuman immunoglobulin molecules by means of a reconstituted or partiallyreconstituted immune system from another animal (see, e.g., publishedPCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741.

A vaccine adjuvant that enhances immune responsiveness to specificantigen.

An adjuvant to enhance tumor-specific immune responses.

An adjuvant to enhance anti-viral immune responses. Anti-viral immuneresponses that may be enhanced using the compositions of the inventionas an adjuvant, include virus and virus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a virus, disease, or symptom selected from the groupconsisting of- AIDS, meningitis, Dengue, EBV, and hepatitis (e.g.,hepatitis B). In another specific embodiment, the compositions of theinvention are used as an adjuvant to enhance an immune response to avirus, disease, or symptom selected from the group consisting ofHIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese Bencephalitis, Influenza A and B, Parainfluenza, Measles,Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpessimplex, and yellow fever.

An adjuvant to enhance anti-bacterial or anti-fungal immune responses.Anti-bacterial or anti-fungal immune responses that may be enhancedusing the compositions of the invention as an adjuvant, include bacteriaor fungus and bacteria or fungus associated diseases or symptomsdescribed herein or otherwise known in the art. In specific embodiments,the compositions of the invention are used as an adjuvant to enhance animmune response to a bacteria or fungus, disease, or symptom selectedfrom the group consisting of. tetanus, Diphtheria, botulism, andmeningitis type B. In another specific embodiment, the compositions ofthe invention are used as an adjuvant to enhance an immune response to abacteria or fungus, disease, or symptom selected from the groupconsisting of: Vibrio cholerae, Mycobactenum leprae, Salmonella typhi,Salmonella paratyphi, Meisseria meningitidis, Streptococcus pneumoniae,Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli,Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium(malaria).

An adjuvant to enhance anti-parasitic immune responses. Anti-parasiticimmune responses that may be enhanced using the compositions of theinvention as an adjuvant, include parasite and parasite associateddiseases or symptoms described herein or otherwise known in the art. Inspecific embodiments, the compositions of the invention are used as anadjuvant to enhance an immune response to a parasite. In anotherspecific embodiment, the compositions of the invention are used as anadjuvant to enhance an immune response to Plasmodium (malaria).

As a stimulator of B cell responsiveness to pathogens.

As an activator of T cells.

As an agent that elevates the immune status of an individual prior totheir receipt of immunosuppressive therapies.

As an agent to induce higher affinity antibodies.

As an agent to increase serum immunoglobulin concentrations.

As an agent to accelerate recovery of immunocompromised individuals.

As an agent to boost immunoresponsiveness among aged populations.

As an immune system enhancer prior to, during, or after bone marrowtransplant and/or other transplants (e.g., allogeneic or xenogeneicorgan transplantation). With respect to transplantation, compositions ofthe invention may be administered prior to, concomitant with, and/orafter transplantation. In a specific embodiment, compositions of theinvention are administered after transplantation, prior to the beginningof recovery of T-cell populations. In another specific embodiment,compositions of the invention are first administered aftertransplantation after the beginning of recovery of T cell populations,but prior to full recovery of B cell populations.

As an agent to boost immunoresponsiveness among individuals having anacquired loss of B cell function. Conditions resulting in an acquiredloss of B cell function that may be ameliorated or treated byadministering the polypeptides, antibodies, polynucleotides and/oragonists or antagonists thereof, include, but are not limited to, HIVInfection, AIDS, bone marrow transplant, and B cell chronic lymphocyticleukemia (CLL).

As an agent to boost immunoresponsiveness among individuals having atemporary immune deficiency. Conditions resulting in a temporary immunedeficiency that may be ameliorated or treated by administering thepolypeptides, antibodies, polynucleotides and/or agonists or antagoniststhereof, include, but are not limited to, recovery from viral infections(e.g., influenza), conditions associated with malnutrition, recoveryfrom infectious mononucleosis, or conditions associated with stress,recovery from measles, recovery from blood transfusion, recovery fromsurgery.

As a regulator of antigen presentation by monocytes, dendritic cells,and/or B-cells. In one embodiment, polynucleotides, polypeptides,antibodies, and/or agonists or antagonists of the present inventionenhance antigen presentation or antagonizes antigen presentation invitro or in vivo. Moreover, in related embodiments, said enhancement orantagonization of antigen presentation may be useful as an anti-tumortreatment or to modulate the immune system.

As an agent to direct an individuals immune system towards developmentof a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

As a means to induce tumor proliferation and thus make it moresusceptible to anti-neoplastic agents. For example, multiple myeloma isa slowly dividing disease and is thus refractory to virtually allanti-neoplastic regimens. If these cells were forced to proliferate morerapidly their susceptibility profile would likely change.

As a stimulator of B cell production in pathologies such as AIDS,chronic lymphocyte disorder and/or Common Variable Immunodificiency.

As a therapy for generation and/or regeneration of lymphoid tissuesfollowing surgery, trauma or genetic defect.

As a gene-based therapy for genetically inherited disorders resulting inimmuno-incompetence such as observed among SCID patients.

As an antigen for the generation of antibodies to inhibit or enhanceimmune mediated responses against polypeptides of the invention.

As a means of activating T cells.

As a means of activating monocytes/macrophages to defend againstparasitic diseases that effect monocytes such as Leshmania.

As pretreatment of bone marrow samples prior to transplant. Suchtreatment would increase B cell representation and thus acceleraterecover.

As a means of regulating secreted cytokines that are elicited bypolypeptides of the invention.

Additionally, polypeptides or polynucleotides of the invention, and/oragonists thereof, may be used to treat or prevent IgE-mediated allergicreactions. Such allergic reactions include, but are not limited to,asthma, rhinitis, and eczema.

All of the above described applications as they may apply to veterinarymedicine.

Antagonists of the invention include, for example, binding and/orinhibitory antibodies, antisense nucleic acids, ribozymes or solubleforms of the secreted-like receptor(s) (e.g., a secreted-like-Fc fusionprotein) (see e.g., Example 9). These would be expected to reverse manyof the activities of the ligand described above as well as find clinicalor practical application as:

A means of blocking various aspects of immune responses to foreignagents or self Examples include autoimmune disorders such as lupus, andarthritis, as well as immunoresponsiveness to skin allergies,inflammation, bowel disease, injury and pathogens.

A therapy for preventing the B cell proliferation and Ig secretionassociated with autoimmune diseases such as idiopathic thrombocytopenicpurpura, systemic lupus erythramatosus and MS.

An inhibitor of B and/or T cell migration in endothelial cells. Thisactivity disrupts tissue architecture or cognate responses and isuseful, for example in disrupting immune responses, and blocking sepsis.

An inhibitor of graft versus host disease or transplant rejection.

A therapy for B cell and/or T cell malignancies such as ALL, Hodgkinsdisease, non-Hodgkins lymphoma, Chronic lymphocyte leukemia,plasmacytomas, multiple myeloma, Burkitt's lymphoma, and EBV-transfomeddiseases.

A therapy for chronic hypergammaglobulinemeia evident in such diseasesas monoclonalgammopathy of undetermined significance (MGUS),Waldenstrom's disease, related idiopathic monoclonalgammopathies, andplasmacytomas.

A therapy for decreasing cellular proliferation of Large B-cellLymphomas.

A means of decreasing the involvement of B cells and Ig associated withChronic Myelogenous Leukemia.

An immunosuppressive agent(s).

Polynucleotides, polypeptides, antibodies, and/or agonists orantagonists of the present invention may be used to modulate IgEconcentrations in vitro or in vivo.

In another embodiment, administration of polypeptides, antibodies,polynucleotides and/or agonists or antagonists of the invention, may beused to treat or prevent IgE-mediated allergic reactions including, butnot limited to, astluna, rhinitis, and eczema.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described herein.

The agonists or antagonists may be employed for instance to inhibitpolypeptide chemotaxis and activation of macrophages and theirprecursors, and of neutrophils, basophils, B lymphocytes and some T-cellsubsets, e.g., activated and CD8 cytotoxic T cells and natural killercells, in certain auto-immune and chronic inflammatory and infectivediseases. Examples of autoimmune diseases are described herein andinclude multiple sclerosis, and insulin-dependent diabetes. Theantagonists or agonists may also be employed to treat infectiousdiseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosisby, for example, preventing the recruitment and activation ofmononuclear phagocytes. They may also be employed to treat idiopathichyper-eosinophilic syndrome by, for example, preventing eosinophilproduction and migration. The antagonists or agonists or may also beemployed for treating atherosclerosis, for example, by preventingmonocyte infiltration in the artery wall.

Antibodies against polypeptides of the invention may be employed totreat ARDS.

Agonists and/or antagonists of the invention also have uses instimulating wound and tissue repair, stimulating angiogenesis,stimulating the repair of vascular or lymphatic diseases or disorders.Additionally, agonists and antagonists of the invention may be used tostimulate the regeneration of mucosal surfaces.

In a specific embodiment, polynucleotides or polypeptides, and/oragonists thereof are used to treat or prevent a disorder characterizedby primary or acquired immunodeficiency, deficient serum immunoglobulinproduction, recurrent infections, and/or immune system dysfunction.Moreover, polynucleotides or polypeptides, and/or agonists thereof maybe used to treat or prevent infections of the joints, bones, skin,and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis,septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g.,those disclosed herein), inflammatory disorders, and malignancies,and/or any disease or disorder or condition associated with theseinfections, diseases, disorders and/or malignancies) including, but notlimited to, CVID, other primary immune deficiencies, HIV disease, CLL,recurrent bronchitis, sinusitis, otitis media, conjunctivitis,pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpeszoster), and/or pneumocystis camii.

In another embodiment, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention are used to treat,and/or diagnose an individual having common variable immunodeficiencydisease (“CVID”; also known as “acquired agammaglobulinemia” and“acquired hypogammaglobulinemia”) or a subset of this disease.

In a specific embodiment, polynucleotides, polypeptides, antibodies,and/or agonists or antagonists of the present invention may be used totreat, diagnose, and/or prevent (1) cancers or neoplasms and (2)autoimmune cell or tissue-related cancers or neoplasms. In a preferredembodiment, polynucleotides, polypeptides, antibodies, and/or agonistsor antagonists of the present invention conjugated to a toxin or aradioactive isotope, as described herein, may be used to treat,diagnose, and/or prevent acute myelogeneous leukemia. In a furtherpreferred embodiment, polynucleotides, polypeptides, antibodies, and/oragonists or antagonists of the present invention conjugated to a toxinor a radioactive isotope, as described herein, may be used to treat,diagnose, and/or prevent, chronic myelogeneous leukemia, multiplemyeloma, non-Hodgkins lymphoma, and/or Hodgkins disease.

In another specific embodiment, polynucleotides or polypeptides, and/oragonists or antagonists of the invention may be used to treat, diagnose,prognose, and/or prevent selective IgA deficiency, myeloperoxidasedeficiency, C2 deficiency, ataxia-telangiectasia, DiGeorge anomaly,common variable immunodeficiency (CVI), X-linked agammaglobulinemia,severe combined immunodeficiency (SCID), chronic granulomatous disease(CGD), and Wiskott-Aldrich syndrome.

Examples of autoimmune disorders that can be treated or detected aredescribed above and also include, but are not limited to: Addison'sDisease, hemolytic anemia, antiphospholipid syndrome, rheumatoidarthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, and/or diagnosed using secreted-like antibodiesand/or anti-secreted-like antibodies and/or a soluble secreted-likepolypeptide of the invention.

In specific embodiments, the compositions of the invention are used asan agent to boost immunoresponsiveness among B cell immunodeficientindividuals, such as, for example, an individual who has undergone apartial or complete splenectomy.

Additionally, polynucleotides, polypeptides, and/or antagonists of theinvention may affect apoptosis, and therefore, would be useful intreating a number of diseases associated with increased cell survival orthe inhibition of apoptosis. For example, diseases associated withincreased cell survival or the inhibition of apoptosis that could betreated or detected by polynucleotides, polypeptides, and/or antagonistsof the invention, include cancers (such as follicular lymphomas,carcinomas with p53 mutations, and hormone-dependent tumors, including,but not limited to colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. In preferred embodiments,polynucleotides, polypeptides, and/or antagonists of the invention areused to inhibit growth, progression, and/or metastisis of cancers, inparticular those listed above.

Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by polynucleotides,polypeptides, and/or antagonists of the invention, include, but are notlimited to, progression, and/or metastases of malignancies and relateddisorders such as leukemia (including acute leukemias (e.g., acutelymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myclocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis that could be treated ordetected by polynucleotides, polypeptides, and/or antagonists of theinvention, include AIDS; neurodegenerative disorders (such asAlzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis,Retinitis pigmentosa, Cerebellar degeneration and brain tumor or priorassociated disease); autoimmune disorders (such as, multiple sclerosis,Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet'sdisease, Crohn's disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis)myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (e.g., hepatitis relatedliver injury, ischemialreperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

Hyperproliferative diseases and/or disorders that could be detectedand/or treated by polynucleotides, polypeptides, and/or antagonists ofthe invention, include, but are not limited to neoplasms located in the:liver, abdomen, bone, breast, digestive system, pancreas, peritoneum,endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary,thymus, thyroid), eye, head and neck, nervous (central and peripheral),lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, andurogenital.

Similarly, other hyperproliferative disorders can also be treated ordetected by polynucleotides, polypeptides, and/or antagonists of theinvention. Examples of such hyperproliferative disorders include, butare not limited to: hypergammaglobulinemia, lymphoproliferativedisorders, paraproteinemias, purpura, sarcoidosis, Sezary Syndrome,Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, andany other hyperproliferative disease, besides neoplasia, located in anorgan system listed above.

Hyperproliferative Disorders

A polynucleotides or polypeptides, or agonists or antagonists of theinvention can be used to treat or detect hyperproliferative disorders,including neoplasms. A polynucleotides or polypeptides, or agonists orantagonists of the present invention may inhibit the proliferation ofthe disorder through direct or indirect interactions. Alliteratively, apolynucleotides or polypeptides, or agonists or antagonists of thepresent invention may proliferate other cells which can inhibit thehyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated. This immune response may beincreased by either enhancing an existing immune response, or byinitiating a new immune response. Alternatively, decreasing an immuneresponse may also be a method of treating hyperproliferative disorders,such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated or detectedby a polynucleotides or polypeptides, or agonists or antagonists of thepresent invention include, but are not limited to neoplasms located inthe: abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvic, skin, soft tissue, spleen,thoracic, and urogenital.

Similarly, other hyperproliferative disorders can also be treated ordetected by a polynucleotides or polypeptides, or agonists orantagonists of the present invention. Examples of suchhyperproliferative disorders include, but are not limited to:hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,Gaucher's Disease, histiocytosis, and any other hyperproliferativedisease, besides neoplasia, located in an organ system listed above.

One preferred embodiment utilizes polynucleotides of the presentinvention to inhibit aberrant cellular division, by gene therapy usingthe present invention, and/or protein fusions or fragments thereof.

Thus, the present invention provides a method for treating cellproliferative disorders by inserting into an abnormally proliferatingcell a polynucleotide of the present invention, wherein saidpolynucleotide represses said expression.

Another embodiment of the present invention provides a method oftreating cell-proliferative disorders in individuals comprisingadministration of one or more active gene copies of the presentinvention to an abnormally proliferating cell or cells. In a preferredembodiment, polynucleotides of the present invention is a DNA constructcomprising a recombinant expression vector effective in expressing a DNAsequence encoding said polynucleotides. In another preferred embodimentof the present invention, the DNA construct encoding the poynucleotidesof the present invention is inserted into cells to be treated utilizinga retrovirus, or more preferably an adenoviral vector (See G J. Nabel,et. al., PNAS 1999 96:324-326, which is hereby incorporated byreference). In a most preferred embodiment, the viral vector isdefective and will not transform non-proliferating cells, onlyproliferating cells. Moreover, in a preferred embodiment, thepolynucleotides of the present invention inserted into proliferatingcells either alone, or in combination with or fused to otherpolynucleotides, can then be modulated via an external stimulus (i.e.magnetic, specific small molecule, chemical, or drug administration,etc.), which acts upon the promoter upstream of said polynucleotides toinduce expression of the encoded protein product. As such the beneficialtherapeutic affect of the present invention may be expressly modulated(i.e. to increase, decrease, or inhibit expression of the presentinvention) based upon said external stimulus.

Polynucleotides of the present invention may be useful in repressingexpression of oncogenic genes or antigens. By “repressing expression ofthe oncogenic genes” is intended the suppression of the transcription ofthe gene, the degradation of the gene transcript (pre-message RNA), theinhibition of splicing, the destruction of the messenger RNA, theprevention of the post-translational modifications of the protein, thedestruction of the protein, or the inhibition of the normal function ofthe protein.

For local administration to abnormally proliferating cells,polynucleotides of the present invention may be administered by anymethod known to those of skill in the art including, but not limited totransfection, electroporation, microinjection of cells, or in vehiclessuch as liposomes, lipofectin, or as naked polynucleotides, or any othermethod described throughout the specification. The polynucleotide of thepresent invention may be delivered by known gene delivery systems suchas, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845(1982); Hocke, Nature 320:275 (1986); Wilson, et al., Proc. Natl. Acad.Sci. U.S.A. 85:3014), vaccinia virus system (Chakrabarty et al., Mol.Cell Biol. 5:3403 (1985) or other efficient DNA delivery systems (Yateset al., Nature 313:812 (1985)) known to those skilled in the art. Thesereferences are exemplary only and are hereby incorporated by reference.In order to specifically deliver or transfect cells which are abnormallyproliferating and spare non-dividing cells, it is preferable to utilizea retrovirus, or adenoviral (as described in the art and elsewhereherein) delivery system known to those of skill in the art. Since hostDNA replication is required for retroviral DNA to integrate and theretrovirus will be unable to self replicate due to the lack of theretrovirus genes needed for its life cycle. Utilizing such a retroviraldelivery system for polynucleotides of the present invention will targetsaid gene and constructs to abnormally proliferating cells and willspare the non-dividing normal cells.

The polynucleotides of the present invention may be delivered directlyto cell proliferative disorder/disease sites in internal organs, bodycavities and the like by use of imaging devices used to guide aninjecting needle directly to the disease site. The polynucleotides ofthe present invention may also be administered to disease sites at thetime of surgical intervention.

By “cell proliferative disease” is meant any human or animal disease ordisorder, affecting any one or any combination of organs, cavities, orbody parts, which is characterized by single or multiple local abnormalproliferations of cells, groups of cells, or tissues, whether benign ormalignant.

Any amount of the polynucleotides of the present invention may beadministered as long as it has a biologically inhibiting effect on theproliferation of the treated cells. Moreover, it is possible toadminister more than one of the polynucleotide of the present inventionsimultaneously to the same site. By “biologically inhibiting” is meantpartial or total growth inhibition as well as decreases in the rate ofproliferation or growth of the cells. The biologically inhibitory dosemay be determined by assessing the effects of the polynucleotides of thepresent invention on target malignant or abnormally proliferating cellgrowth in tissue culture, tumor growth in animals and cell cultures, orany other method known to one of ordinary skill in the art.

The present invention is further directed to antibody-based therapieswhich involve administering of anti-polypeptides and anti-polynucleotideantibodies to a mammalian, preferably human, patient for treating one ormore of the described disorders. Methods for producing anti-polypeptidesand anti-polynucleotide antibodies polyclonal and monoclonal antibodiesare described in detail elsewhere herein. Such antibodies may beprovided in pharmaceutically acceptable compositions as known in the artor as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

In particular, the antibodies, fragments and derivatives of the presentinvention are useful for treating a subject having or developing cellproliferative and/or differentiation disorders as described herein. Suchtreatment comprises administering a single or multiple doses of theantibody, or a fragment, derivative, or a conjugate thereof.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors, for example, which serve toincrease the number or activity of effector cells which interact withthe antibodies.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides,including fragments thereof. Preferred binding affinities include thosewith a dissociation constant or Kd less than 5×10⁻⁶M, 10⁻⁶M, 5×10⁻⁷M,10⁻⁷M, 5×10⁻⁸M, 10⁻⁸M, 5×10⁻⁹M, 10⁻⁹M, 5×10⁻¹⁰M, 10⁻¹⁰M, 5×10⁻¹¹M,10⁻¹¹M, 5×10⁻¹²M, 10⁻¹²M, 5×10⁻¹³M, 10⁻¹³M, 5×10⁻¹⁴M, 10⁻¹⁴M, 5×10⁻¹⁵M,and 10⁻¹⁵M.

Moreover, polypeptides of the present invention are useful in inhibitingthe angiogenesis of proliferative cells or tissues, either alone, as aprotein fusion, or in combination with other polypeptides directly orindirectly, as described elsewhere herein. In a most preferredembodiment, said anti-angiogenesis effect may be achieved indirectly,for example, through the inhibition of hematopoietic, tumor-specificcells, such as tumor-associated macrophages (See Joseph IB, et al. JNatl Cancer Inst, 90(21):1648-53 (1998), which is hereby incorporated byreference). Antibodies directed to polypeptides or polynucleotides ofthe present invention may also result in inhibition of angiogenesisdirectly, or indirectly (See Witte L, et al., Cancer Metastasis Rev.17(2):155-61 (1998), which is hereby incorporated by reference)).

Polypeptides, including protein fusions, of the present invention, orfragments thereof may be useful in inhibiting proliferative cells ortissues through the induction of apoptosis. Said polypeptides may acteither directly, or indirectly to induce apoptosis of proliferativecells and tissues, for example in the activation of a death-domainreceptor, such as tumor necrosis factor (TNF) receptor-1, CD95(Fas/APO-1), TNF-receptor-related apoptosis-mediated protein (TRAMP) andTNF-related apoptosis-inducing ligand (TRAIL) receptor-1 and -2 (SeeSchulze-Osthoff K, et.al., Eur J Biochem 254(3):439-59 (1998), which ishereby incorporated by reference). Moreover, in another preferredembodiment of the present invention, said polypeptides may induceapoptosis through other mechanisms, such as in the activation of otherproteins which will activate apoptosis, or through stimulating theexpression of said proteins, either alone or in combination with smallmolecule drugs or adjuviants, such as apoptonin, galectins,thioredoxins, antiinflammatory proteins (See for example, Mutat Res400(1-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem BiolInteract. April 24;111-112:23-34 (1998), J Mol Med. 76(6):402-12 (1998),Int J Tissue React; 20(1):3-15 (1998), which are all hereby incorporatedby reference).

Polypeptides, including protein fusions to, or fragments thereof, of thepresent invention are useful in inhibiting the metastasis ofproliferative cells or tissues. Inhibition may occur as a direct resultof administering polypeptides, or antibodies directed to saidpolypeptides as described elsewere herein, or indirectly, such asactivating the expression of proteins known to inhibit metastasis, forexample alpha 4 integrins, (See, e.g., Curr Top Microbiol Immunol1998;231:125-41, which is hereby incorporated by reference). Suchtherapeutic affects of the present invention may be achieved eitheralone, or in combination with small molecule drugs or adjuvants.

In another embodiment, the invention provides a method of deliveringcompositions containing the polypeptides of the invention (e.g.,compositions containing polypeptides or polypeptide antibodes associatedwith heterologous polypeptides, heterologous nucleic acids, toxins, orprodrugs) to targeted cells expressing the polypeptide of the presentinvention. Polypeptides or polypeptide antibodes of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions.

Polypeptides, protein fusions to, or fragments thereof, of the presentinvention are useful in enhancing the immunogenicity and/or antigenicityof proliferating cells or tissues, either directly, such as would occurif the polypeptides of the present invention ‘vaccinated’ the immuneresponse to respond to proliferative antigens and immunogens, orindirectly, such as in activating the expression of proteins known toenhance the immune response (e.g. chemokines), to said antigens andimmunogens.

Cardiovascular Disorders

Polynucleotides or polypeptides, or agonists or antagonists of theinvention may be used to treat cardiovascular disorders, includingperipheral artery disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects.

Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardiumn, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stealsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, artenitis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

Polynucleotides or polypeptides, or agonists or antagonists of theinvention, are especially effective for the treatment of critical limbischemia and coronary disease.

Polypeptides may be administered using any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,biolistic injectors, particle accelerators, gelfoam sponge depots, othercommercially available depot materials, osmotic pumps, oral orsuppositorial solid pharmaceutical formulations, decanting or topicalapplications during surgery, aerosol delivery. Such methods are known inthe art. Polypeptides of the invention may be administered as part of aTherapeutic, described in more detail below. Methods of deliveringpolynucleotides of the invention are described in more detail herein.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

The present invention provides for treatment of diseases or disordersassociated with neovascularization by administration of thepolynucleotides and/or polypeptides of the invention, as well asagonists or antagonists of the present invention. Malignant andmetastatic conditions which can be treated with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincon Co., Philadelphia(1985)). Thus, the present invention provides a method of treating anangiogenesis-related disease and/or disorder, comprising administeringto an individual in need thereof a therapeutically effective amount of apolynucleotide, polypeptide, antagonist and/or agonist of the invention.For example, polynucleotides, polypeptides, antagonists and/or agonistsmay be utilized in a variety of additional methods in order totherapeutically treat a cancer or tumor. Cancers which may be treatedwith polynucleotides, polypeptides, antagonists and/or agonists include,but are not limited to solid tumors, including prostate, lung, breast,ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,bladder, thyroid cancer; primary tumors and metastases; melanomas;glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non-small cell lungcancer; colorectal cancer; advanced malignancies; and blood born tumorssuch as leukemias. For example, polynucleotides, polypeptides,antagonists and/or agonists may be delivered topically, in order totreat cancers such as skin cancer, head and neck tumors, breast tumors,and Kaposi's sarcoma.

Within yet other aspects, polynucleotides, polypeptides, antagonistsand/or agonists may be utilized to treat superficial forms of bladdercancer by, for example, intravesical administration. Polynucleotides,polypeptides, antagonists and/or agonists may be delivered directly intothe tumor, or near the tumor site, via injection or a catheter. Ofcourse, as the artisan of ordinary skill will appreciate, theappropriate mode of administration will vary according to the cancer tobe treated. Other modes of delivery are discussed herein.

Polynucleotides, polypeptides, antagonists and/or agonists may be usefulin treating other disorders, besides cancers, which involveangiogenesis. These disorders include, but are not limited to: benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas; artheroscleric plaques; ocularangiogenic diseases, for example, diabetic retinopathy, retinopathy ofprematurity, macular degeneration, corneal graft rejection, neovascularglaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis andPterygia (abnormal blood vessel growth) of the eye; rheumatoidarthritis; psoriasis; delayed wound healing; endometriosis;vasculogenesis; granulations; hypertrophic scars (keloids); nonunionfractures; scleroderma; trachoma; vascular adhesions; myocardialangiogenesis; coronary collaterals; cerebral collaterals; arteriovenousmalformations; ischemic limb angiogenesis; Osler-Webber Syndrome; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;fibromuscular dysplasia; wound granulation; Crohn's disease; andatherosclerosis.

For example, within one aspect of the present invention methods areprovided for treating hypertrophic scars and keloids, comprising thestep of administering a polynucleotide, polypeptide, antagonist and/oragonist of the invention to a hypertrophic scar or keloid.

Within one embodiment of the present invention polynucleotides,polypeptides, antagonists and/or agonists are directly injected into ahypertrophic scar or keloid, in order to prevent the progression ofthese lesions. This therapy is of particular value in the prophylactictreatment of conditions which are known to result in the development ofhypertrophic scars and keloids (e.g., bums), and is preferably initiatedafter the proliferative phase has had time to progress (approximately 14days after the initial injury), but before hypertrophic scar or keloiddevelopment. As noted above, the present invention also provides methodsfor treating neovascular diseases of the eye, including for example,corneal neovascularization, neovascular glaucoma, proliferative diabeticretinopathy, retrolental fibroplasia and macular degeneration.

Moreover, Ocular disorders associated with neovascularization which canbe treated with the polynucleotides and polypeptides of the presentinvention (including agonists and/or antagonists) include, but are notlimited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma,retrolental fibroplasia, uveitis, retinopathy of prematurity maculardegeneration, corneal graft neovascularization, as well as other eyeinflammatory diseases, ocular tumors and diseases associated withchoroidal or iris neovascularization. See, e.g., reviews by Waltman etal., Am. J. Ophthal. 85:704-710 (1978) and Gartner et al., Surv.Ophthal. 22:291-312 (1978).

Thus, within one aspect of the present invention methods are providedfor treating neovascular diseases of the eye such as cornealneovascularization (including corneal graft neovascularization),comprising the step of administering to a patient a therapeuticallyeffective amount of a compound (as described above) to the cornea, suchthat the formation of blood vessels is inhibited. Briefly, the cornea isa tissue which normally lacks blood vessels. In certain pathologicalconditions however, capillaries may extend into the cornea from thepericorneal vascular plexus of the limbus. When the cornea becomesvascularized, it also becomes clouded, resulting in a decline in thepatient's visual acuity. Visual loss may become complete if the corneacompletely opacitates. A wide variety of disorders can result in cornealneovascularization, including for example, corneal infections (e.g.,trachoma, herpes simplex keratitis, leishmaniasis and onchocerciasis),immunological processes (e.g., graft rejection and Stevens-Johnson'ssyndrome), alkali bums, trauma, inflammation (of any cause), toxic andnutritional deficiency states, and as a complication of wearing contactlenses.

Within particularly preferred embodiments of the invention, may beprepared for topical administration in saline (combined with any of thepreservatives and antimicrobial agents commonly used in ocularpreparations), and administered in eyedrop form. The solution orsuspension may be prepared in its pure form and administered severaltimes daily. Alternatively, anti-angiogenic compositions, prepared asdescribed above, may also be administered directly to the cornea. Withinpreferred embodiments, the anti-angiogenic composition is prepared witha muco-adhesive polymer which binds to cornea. Within furtherembodiments, the anti-angiogenic factors or anti-angiogenic compositionsmay be utilized as an adjunct to conventional steroid therapy. Topicaltherapy may also be useful prophylactically in corneal lesions which areknown to have a high probability of inducing an angiogenic response(such as chemical bums). In these instances the treatment, likely incombination with steroids, may be instituted immediately to help preventsubsequent complications.

Within other embodiments, the compounds described above may be injecteddirectly into the corneal stroma by an ophthalmologist under microscopicguidance. The preferred site of injection may vary with the morphologyof the individual lesion, but the goal of the administration would be toplace the composition at the advancing front of the vasculature (i.e.,interspersed between the blood vessels and the normal cornea). In mostcases this would involve perilimbic corneal injection to “protect” thecornea from the advancing blood vessels. This method may also beutilized shortly after a corneal insult in order to prophylacticallyprevent corneal neovascularization. In this situation the material couldbe injected in the perilimbic cornea interspersed between the corneallesion and its undesired potential limbic blood supply. Such methods mayalso be utilized in a similar fashion to prevent capillary invasion oftransplanted corneas. In a sustained-release form injections might onlybe required 2-3 times per year. A steroid could also be added to theinjection solution to reduce inflammation resulting from the injectionitself.

Within another aspect of the present invention, methods are provided fortreating neovascular glaucoma, comprising the step of administering to apatient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eye, such that theformation of blood vessels is inhibited. In one embodiment, the compoundmay be administered topically to the eye in order to treat early formsof neovascular glaucoma. Within other embodiments, the compound may beimplanted by injection into the region of the anterior chamber angle.Within other embodiments, the compound may also be placed in anylocation such that the compound is continuously released into theaqueous humor. Within another aspect of the present invention, methodsare provided for treating proliferative diabetic retinopathy, comprisingthe step of administering to a patient a therapeutically effectiveamount of a polynucleotide, polypeptide, antagonist and/or agonist tothe eyes, such that the formation of blood vessels is inhibited.

Within particularly preferred embodiments of the invention,proliferative diabetic retinopathy may be treated by injection into theaqueous humor or the vitreous, in order to increase the localconcentration of the polynucleotide, polypeptide, antagonist and/oragonist in the retina. Preferably, this treatment should be initiatedprior to the acquisition of severe disease requiring photocoagulation.

Within another aspect of the present invention, methods are provided fortreating retrolental fibroplasia, comprising the step of administeringto a patient a therapeutically effective amount of a polynucleotide,polypeptide, antagonist and/or agonist to the eye, such that theformation of blood vessels is inhibited. The compound may beadministered topically, via intravitreous injection and/or viaintraocular implants.

Additionally, disorders which can be treated with the polynucleotides,polypeptides, agonists and/or agonists include, but are not limited to,hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques,delayed wound healing, granulations, hemophilic joints, hypertrophicscars, nonunion fractures, Osler-Weber syndrome, pyogenic granuloma,scleroderma, trachoma, and vascular adhesions.

Moreover, disorders and/or states, which can be treated with be treatedwith the polynucleotides, polypeptides, agonists and/or agonistsinclude, but are not limited to, solid tumors, blood born tumors such asleukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, forexample hemangiomas, acoustic neuromas, neurofibromas, trachomas, andpyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenicdiseases, for example, diabetic retinopathy, retinopathy of prematurity,macular degeneration, corneal graft rejection, neovascular glaucoma,retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayedwound healing, endometriosis, vascluogenesis, granulations, hypertrophicscars (keloids), nonunion fractures, scleroderma, trachoma, vascularadhesions, myocardial angiogenesis, coronary collaterals, cerebralcollaterals, arteriovenous malformations, ischemic limb angiogenesis,Osler-Webber Syndrome, plaque neovascularization, telangiectasia,hemophiliac joints, angiofibroma fibromuscular dysplasia, woundgranulation, Crohn's disease, atherosclerosis, birth control agent bypreventing vascularization required for embryo implantation controllingmenstruation, diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa),ulcers (Helicobacter pylori), Bartonellosis and bacillary angiomatosis.

In one aspect of the birth control method, an amount of the compoundsufficient to block embryo implantation is administered before or afterintercourse and fertilization have occurred, thus providing an effectivemethod of birth control, possibly a “morning after” method.Polynucleotides, polypeptides, agonists and/or agonists may also be usedin controlling menstruation or administered as either a peritoneallavage fluid or for peritoneal implantation in the treatment ofendometriosis.

Polynucleotides, polypeptides, agonists and/or agonists of the presentinvention may be incorporated into surgical sutures in order to preventstitch granulomas.

Polynucleotides, polypeptides, agonists and/or agonists may be utilizedin a wide variety of surgical procedures. For example, within one aspectof the present invention a compositions (in the form of, for example, aspray or film) may be utilized to coat or spray an area prior to removalof a tumor, in order to isolate normal surrounding tissues frommalignant tissue, and/or to prevent the spread of disease to surroundingtissues. Within other aspects of the present invention, compositions(e.g., in the form of a spray) may be delivered via endoscopicprocedures in order to coat tumors, or inhibit angiogenesis in a desiredlocale. Within yet other aspects of the present invention, surgicalmeshes which have been coated with anti- angiogenic compositions of thepresent invention may be utilized in any procedure wherein a surgicalmesh might be utilized. For example, within one embodiment of theinvention a surgical mesh laden with an anti-angiogenic composition maybe utilized during abdominal cancer resection surgery (e.g., subsequentto colon resection) in order to provide support to the structure, and torelease an amount of the anti-angiogenic factor.

Within further aspects of the present invention, methods are providedfor treating tumor excision sites, comprising administering apolynucleotide, polypeptide, agonist and/or agonist to the resectionmargins of a tumor subsequent to excision, such that the localrecurrence of cancer and the formation of new blood vessels at the siteis inhibited. Within one embodiment of the invention, theanti-angiogenic compound is administered directly to the tumor excisionsite (e.g., applied by swabbing, brushing or otherwise coating theresection margins of the tumor with the anti-angiogenic compound).Alliteratively, the anti-angiogenic compounds may be incorporated intoknown surgical pastes prior to administration. Within particularlypreferred embodiments of the invention, the anti-angiogenic compoundsare applied after hepatic resections for malignancy, and afterneurosurgical operations.

Within one aspect of the present invention, polynucleotides,polypeptides, agonists and/or agonists may be administered to theresection margin of a wide variety of tumors, including for example,breast, colon, brain and hepatic tumors. For example, within oneembodiment of the invention, anti-angiogenic compounds may beadministered to the site of a neurological tumor subsequent to excision,such that the formation of new blood vessels at the site are inhibited.

The polynucleotides, polypeptides, agonists and/or agonists of thepresent invention may also be administered along with otheranti-angiogenic factors. Representative examples of otheranti-angiogenic factors include: Anti-Invasive Factor, retinoic acid andderivatives thereof, paclitaxel, Suramin, Tissue Inhibitor ofMetalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2,Plasminogen Activator Inhibitor-1, Plasminogen Activator Inhibitor-2,and various forms of the lighter “d group” transition metals.

Lighter “d group” transition metals include, for example, vanadium,molybdenum, tungsten, titanium, niobium, and tantalum species. Suchtransition metal species may form transition metal complexes. Suitablecomplexes of the above-mentioned transition metal species include oxotransition metal complexes.

Representative examples of vanadium complexes include oxo vanadiumcomplexes such as vanadate and vanadyl complexes. Suitable vanadatecomplexes include metavanadate and orthovanadate complexes such as, forexample, ammonium metavanadate, sodium metavanadate, and sodiumorthovanadate. Suitable vanadyl complexes include, for example, vanadylacetylacetonate and vanadyl sulfate including vanadyl sulfate hydratessuch as vanadyl sulfate mono- and trihydrates.

Representative examples of tungsten and molybdenum complexes alsoinclude oxo complexes. Suitable oxo tungsten complexes include tungstateand tungsten oxide complexes. Suitable tungstate complexes includeammonium tungstate, calcium tungstate, sodium tungstate dihydrate, andtungstic acid. Suitable tungsten oxides include tungsten (IV) oxide andtungsten (VI) oxide. Suitable oxo molybdenum complexes includemolybdate, molybdenum oxide, and molybdenyl complexes. Suitablemolybdate complexes include ammonium molybdate and its hydrates, sodiummolybdate and its hydrates, and potassium molybdate and its hydrates.Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum(VI) oxide, and molybdic acid. Suitable molybdenyl complexes include,for example, molybdenyl acetylacetonate. Other suitable tungsten andmolybdenum complexes include hydroxo derivatives derived from, forexample, glycerol, tartaric acid, and sugars.

A wide variety of other anti-angiogenic factors may also be utilizedwithin the context of the present invention. Representative examplesinclude platelet factor 4; protamine sulphate; sulphated chitinderivatives (prepared from queen crab shells), (Murata et al., CancerRes. 51:22-26, 1991); Sulphated Polysaccharide Peptidoglycan Complex(SP-PG) (the function of this compound may be enhanced by the presenceof steroids such as estrogen, and tamoxifen citrate); Staurosporine;modulators of matrix metabolism, including for example, proline analogs,cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline,alpha,alpha-dipyridyl, aminopropionitrile fumarate;4-propyl-5-(4-pyridinyl)-2(3H)-oxazolone; Methotrexate; Mitoxantrone;Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al., J.Bio. Chem. 267:17321-17326, 1992); Chymostatin (Tomkinson et al.,Biochem J. 286:475-480, 1992); Cyclodextrin Tetradecasulfate;Eponemycin; Camptothecin; Fumagillin (Ingber et al., Nature 348:555-557,1990); Gold Sodium Thiomalate (“GST”; Matsubara and Ziff, J. Clin.Invest. 79:1440-1446, 1987); anticollagenase-serum; alpha2-antiplasmin(Holmes et al., J. Biol. Chem. 262(4):1659-1664, 1987); Bisantrene(National Cancer Institute); Lobenzarit disodium(N-(2)-carboxyphenyl4-chloroanthronilic acid disodium or “CCA”; Takeuchiet al., Agents Actions 36:312-316, 1992); Thalidomide; Angostaticsteroid; AGM-1470; carboxynaminolmidazole; and metalloproteinaseinhibitors such as BB94.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated or detected by the polynucleotides orpolypeptides and/or antagonists or agonists of the invention, includecancers (such as follicular lymphomas, carcinomas with p53 mutations,and hormone-dependent tumors, including, but not limited to coloncancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma,glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomachcancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma,osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome,Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn'sdisease, polymyositis, systemic lupus erythematosus and immune-relatedglomerulonephritis and rheumatoid arthritis) and viral infections (suchas herpes viruses, pox viruses and adenoviruses), inflammation, graft v.host disease, acute graft rejection, and chronic graft rejection. Inpreferred embodiments, the polynucleotides or polypeptides, and/oragonists or antagonists of the invention are used to inhibit growth,progression, and/or metasis of cancers, in particular those listedabove.

Additional diseases or conditions associated with increased cellsurvival that could be treated or detected by the polynucleotides orpolypeptides, or agonists or antagonists of the invention, include, butare not limited to, progression, and/or metastases of malignancies andrelated disorders such as leukemia (including acute leukemias (e.g.,acute lymphocytic leukemia, acute myelocytic leukemia (includingmyeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be treated ordetected by the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, include AIDS; neurodegenerative disorders(such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateralsclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumoror prior associated disease); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.host disease, ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), liver injury (e.g.,hepatitis related liver injury, ischemia/reperfuision injury,cholestosis (bile duct injury) and liver cancer); toxin-induced liverdisease (such as that caused by alcohol), septic shock, cachexia andanorexia.

Neural Activity and Neurological Diseases

The polynucleotides, polypeptides and agonists or antagonists of theinvention may be used for the diagnosis and/or treatment of diseases,disorders, damage or injury of the brain and/or nervous system. Nervoussystem disorders that can be treated with the compositions of theinvention (e.g., secreted polypeptides, polynucleotides, and/or agonistsor antagonists), include, but are not limited to, nervous systeminjuries, and diseases or disorders which result in either adisconnection of axons, a diminution or degeneration of neurons, ordemyelination. Nervous system lesions which may be treated in a patient(including human and non-human mammalian patients) according to themethods of the invention, include but are not limited to, the followinglesions of either the central (including spinal cord, brain) orperipheral nervous systems: (1) ischemic lesions, in which a lack ofoxygen in a portion of the nervous system results in neuronal injury ordeath, including cerebral infarction or ischemia, or spinal cordinfarction or ischemia; (2) traumatic lesions, including lesions causedby physical injury or associated with surgery, for example, lesionswhich sever a portion of the nervous system, or compression injuries;(3) malignant lesions, in which a portion of the nervous system isdestroyed or injured by malignant tissue which is either a nervoussystem associated malignancy or a malignancy derived from non-nervoussystem tissue; (4) infectious lesions, in which a portion of the nervoussystem is destroyed or injured as a result of infection, for example, byan abscess or associated with infection by human immunodeficiency virus,herpes zoster, or herpes simplex virus or with Lyme disease,tuberculosis, or syphilis; (5) degenerative lesions, in which a portionof the nervous system is destroyed or injured as a result of adegenerative process including but not limited to, degenerationassociated with Parkinson's disease, Alzheimer's disease, Huntington'schorea, or amyotrophic lateral sclerosis (ALS); (6) lesions associatedwith nutritional diseases or disorders, in which a portion of thenervous system is destroyed or injured by a nutritional disorder ordisorder of metabolism including, but not limited to, vitamin B12deficiency, folic acid deficiency, Wernicke disease, tobacco-alcoholamblyopia, Marchiafava-Bignami disease (primary degeneration of thecorpus callosum), and alcoholic cerebellar degeneration; (7)neurological lesions associated with systemic diseases including, butnot limited to, diabetes (diabetic neuropathy, Bell's palsy), systemiclupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused bytoxic substances including alcohol, lead, or particular neurotoxins; and(9) demyelinated lesions in which a portion of the nervous system isdestroyed or injured by a demyelinating disease including, but notlimited to, multiple sclerosis, human immunodeficiency virus-associatedmyelopathy, transverse myelopathy or various etiologies, progressivemultifocal leukoencephalopathy, and central pontine myelinolysis.

In one embodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to protect neural cells from thedamaging effects of hypoxia. In a further preferred embodiment, thepolypeptides, polynucleotides, or agonists or antagonists of theinvention are used to protect neural cells from the damaging effects ofcerebral hypoxia. According to this embodiment, the compositions of theinvention are used to treat or prevent neural cell injury associatedwith cerebral hypoxia. In one non-exclusive aspect of this embodiment,the polypeptides, polynucleotides, or agonists or antagonists of theinvention, are used to treat or prevent neural cell injury associatedwith cerebral ischemia. In another non-exclusive aspect of thisembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat or prevent neural cellinjury associated with cerebral infarction.

In another preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat or preventneural cell injury associated with a stroke. In a specific embodiment,the polypeptides, polynucleotides, or agonists or antagonists of theinvention are used to treat or prevent cerebral neural cell injuryassociated with a stroke.

In another preferred embodiment, the polypeptides, polynucleotides, oragonists or antagonists of the invention are used to treat or preventneural cell injury associated with a heart attack. In a specificembodiment, the polypeptides, polynucleotides, or agonists orantagonists of the invention are used to treat or prevent cerebralneural cell injury associated with a heart attack.

The compositions of the invention which are useful for treating orpreventing a nervous system disorder may be selected by testing forbiological activity in promoting the survival or differentiation ofneurons. For example, and not by way of limitation, compositions of theinvention which elicit any of the following effects may be usefulaccording to the invention: (1) increased survival time of neurons inculture either in the presence or absence of hypoxia or hypoxicconditions; (2) increased sprouting of neurons in culture or in vivo;(3) increased production of a neuron-associated molecule in culture orin vivo, e.g., choline acetyltransferase or acetylcholinesterase withrespect to motor neurons; or (4) decreased symptoms of neurondysfunction in vivo. Such effects may be measured by any method known inthe art. In preferred, non-limiting embodiments, increased survival ofneurons may routinely be measured using a method set forth herein orotherwise known in the art, such as, for example, in Zhang et al., ProcNatl Acad Sci USA 97:3637-42 (2000) or in Arakawa et al., J. Neurosci.,10:3507-15 (1990); increased sprouting of neurons may be detected bymethods known in the art, such as, for example, the methods set forth inPestronk et al., Exp. Neurol., 70:65-82 (1980), or Brown et al., Ann.Rev. Neurosci, 4:17-42 (1981): increased production of neuron-associatedmolecules may be measured by bioassay, enzymatic assay, antibodybinding, Northern blot assay, etc., using techniques known in the artand depending on the molecule to be measured; and motor neurondysfunction may be measured by assessing the physical manifestation ofmotor neuron disorder, e.g., weakness, motor neuron conduction velocity,or functional disability.

In specific embodiments, motor neuron disorders that may be treatedaccording to the invention include, but are not limited to, disorderssuch as infarction, infection, exposure to toxin, trauma, surgicaldamage, degenerative disease or malignancy that may affect motor neuronsas well as other components of the nervous system, as well as disordersthat selectively affect neurons such as amyotrophic lateral sclerosis,and including, but not limited to, progressive spinal muscular atrophy,progressive bulbar palsy, primary lateral sclerosis, infantile andjuvenile muscular atrophy, progressive bulbar paralysis of childhood(Fazio-Londe syndrome), poliomyelitis and the post polio syndrome, andHereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).

Further, polypeptides or polynucleotides of the invention may play arole in neuronal survival; synapse formation; conductance; neuraldifferentiation, etc. Thus, compositions of the invention (includingsecreted polynucleotides, polypeptides, and agonists or antagonists) maybe used to diagnose and/or treat or prevent diseases or disordersassociated with these roles, including, but not limited to, learningand/or cognition disorders. The compositions of the invention may alsobe useful in the treatment or prevention of neurodegenerative diseasestates and/or behavioural disorders. Such neurodegenerative diseasestates and/or behavioral disorders include, but are not limited to,Alzheimers Disease, Parkinsons Disease, Huntingtons Disease, TouretteSyndrome, schizophrenia, mania, dementia, paranoia, obsessive compulsivedisorder, panic disorder, learning disabilities, ALS, psychoses, autism,and altered behaviors, including disorders in feeding, sleep patterns,balance, and perception. In addition, compositions of the invention mayalso play a role in the treatment, prevention and/or detection ofdevelopmental disorders associated with the developing embryo, orsexually-linked disorders.

Additionally, polypeptides, polynucleotides and/or agonists orantagonists of the invention, may be useful in protecting neural cellsfrom diseases, damage, disorders, or injury, associated withcerebrovascular disorders including, but not limited to, carotid arterydiseases (e.g., carotid artery thrombosis, carotid stenosis, or MoyamoyaDisease), cerebral amyloid angiopathy, cerebral aneurysm, cerebralanoxia, cerebral arteriosclerosis, cerebral arteriovenous malformations,cerebral artery diseases, cerebral embolism and thrombosis (e.g.,carotid artery thrombosis, sinus thrombosis, or Wallenberg's Syndrome),cerebral hemorrhage (e.g., epidural or subdural hematoma, orsubarachnoid hemorrhage), cerebral infarction, cerebral ischemia (e.g.,transient cerebral ischemia, Subclavian Steal Syndrome, orvertebrobasilar insufficiency), vascular dementia (e.g., multi-infarct),leukomalacia, periventricular, and vascular headache (e.g., clusterheadache or migraines).

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing polynucleotides or polypeptides, aswell as agonists or antagonists of the present invention, fortherapeutic purposes, for example, to stimulate neurological cellproliferation and/or differentiation. Therefore, polynucleotides,polypeptides, agonists and/or antagonists of the invention may be usedto treat and/or detect neurologic diseases. Moreover, polynucleotides orpolypeptides, or agonists or antagonists of the invention, can be usedas a marker or detector of a particular nervous system disease ordisorder.

Examples of neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include brain diseases, such as metabolic braindiseases which includes phenylketonuria such as maternalphenylketonuria, pyruvate carboxylase deficiency, pyruvate dehydrogenasecomplex deficiency, Wemicke's Encephalopathy, brain edema, brainneoplasms such as cerebellar neoplasms which include infratentorialneoplasms, cerebral ventricle neoplasms such as choroid plexusneoplasms, hypothalamic neoplasms, supratentorial neoplasms, canavandisease, cerebellar diseases such as cerebellar ataxia which includespinocerebellar degeneration such as ataxia telangiectasia, cerebellardyssynergia, Friederich's Ataxia, Machado-Joseph Disease,olivopontocerebellar atrophy, cerebellar neoplasms such asinfratentorial neoplasms, diffuse cerebral sclerosis such asencephalitis periaxialis, globoid cell leukodystrophy, metachromaticleukodystrophy and subacute sclerosing panencephalitis.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include cerebrovascular disorders (such as carotidartery diseases which include carotid artery thrombosis, carotidstenosis and Moyamoya Disease), cerebral amyloid angiopathy, cerebralaneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebralarteriovenous malformations, cerebral artery diseases, cerebral embolismand thrombosis such as carotid artery thrombosis, sinus thrombosis andWallenberg's Syndrome, cerebral hemorrhage such as epidural hematoma,subdural hematoma and subarachnoid hemorrhage, cerebral infarction,cerebral ischemia such as transient cerebral ischemia, Subclavian StealSyndrome and vertebrobasilar insufficiency, vascular dementia such asmulti-infarct dementia, periventricular leukomalacia, vascular headachesuch as cluster headache and migraine.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include dementia such as AIDS Dementia Complex,presenile dementia such as Alzheimer's Disease and Creutzfeldt-JakobSyndrome, senile dementia such as Alzheimer's Disease and progressivesupranuclear palsy, vascular dementia such as multi-infarct dementia,encephalitis which include encephalitis periaxialis, viral encephalitissuch as epidemic encephalitis, Japanese Encephalitis, St. LouisEncephalitis, tick-borne encephalitis and West Nile Fever, acutedisseminated encephalomyelitis, meningoencephalitis such asuveomeningoencephalitic syndrome, Postencephalitic Parkinson Disease andsubacute sclerosing panencephalitis, encephalomalacia such asperiventricular leukomalacia, epilepsy such as generalized epilepsywhich includes infantile spasms, absence epilepsy, myoclonic epilepsywhich includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsysuch as complex partial epilepsy, frontal lobe epilepsy and temporallobe epilepsy, post-traumatic epilepsy, status epilepticus such asEpilepsia Partialis Continua, and Hallervorden-Spatz Syndrome.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include hydrocephalus such as Dandy-Walker Syndromeand normal pressure hydrocephalus, hypothalamic diseases such ashypothalamic neoplasms, cerebral malaria, narcolepsy which includescataplexy, bulbar poliomyelitis, cerebri pseudotumor, Rett Syndrome,Reye's Syndrome, thalamic diseases, cerebral toxoplasmosis, intracranialtuberculoma and Zellweger Syndrome, central nervous system infectionssuch as AIDS Dementia Complex, Brain Abscess, subdural empyema,encephalomyelitis such as Equine Encephalomyelitis, Venezuelan EquineEncephalomyelitis, Necrotizing Hemorrhagic Encephalomyelitis, Visna, andcerebral malaria.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include meningitis such as arachnoiditis, asepticmeningtitis such as viral meningtitis which includes lymphocyticchoriomeningitis, Bacterial meningtitis which includes HaemophilusMeningtitis, Listeria Meningtitis, Meningococcal Meningtitis such asWaterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningealtuberculosis, fungal meningitis such as Cryptococcal Meningtitis,subdural effusion, meningoencephalitis such as uvemeningoencephaliticsyndrome, myelitis such as transverse myelitis, neurosyphilis such astabes dorsalis, poliomyelitis which includes bulbar poliomyelitis andpostpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-JakobSyndrome, Bovine Spongiform Encephalopathy, Gerstmann-StrausslerSyndrome, Kuru, Scrapie), and cerebral toxoplasmosis.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include central nervous system neoplasms such as brainneoplasms that include cerebellar neoplasms such as infratentorialneoplasms, cerebral ventricle neoplasms such as choroid plexusneoplasms, hypothalamic neoplasms and supratentorial neoplasms,meningeal neoplasms, spinal cord neoplasms which include epiduralneoplasms, demyelinating diseases such as Canavan Diseases, diffusecerebral sceloris which includes adrenoleukodystrophy, encephalitisperiaxialis, globoid cell leukodystrophy, diffuse cerebral sclerosissuch as metachromatic leukodystrophy, allergic encephalomyelitis,necrotizing hemorrhagic encephalomyelitis, progressive multifocalleukoencephalopathy, multiple sclerosis, central pontine myelinolysis,transverse myelitis, neuromyelitis optica, Scrapie, Swayback ChronicFatigue Syndrome, Visna, High Pressure Nervous Syndrome, Meningism,spinal cord diseases such as amyotonia congenita, amyotrophic lateralsclerosis, spinal muscular atrophy such as Werdnig-Hoffmann Disease,spinal cord compression, spinal cord neoplasms such as epiduralneoplasms, syringomyelia, Tabes Dorsalis, Stiff-Man Syndrome, mentalretardation such as Angelman Syndrome, Cri-du-Chat Syndrome, De Lange'sSyndrome, Down Syndrome, Gangliosidoses such as gangliosidoses G(MI),Sandhoff Disease, Tay-Sachs Disease, Hartnup Disease, homocystinuria,Laurence-Moon- Biedl Syndrome, Lesch-Nyhan Syndrome, Maple Syrup UrineDisease, mucolipidosis such as fucosidosis, neuronalceroid-lipofuscinosis, oculocerebrorenal syndrome, phenylketonuria suchas maternal phenylketonuria, Prader-Willi Syndrome, Rett Syndrome,Rubinstein-Taybi Syndrome, Tuberous Sclerosis, WAGR Syndrome, nervoussystem abnormalities such as holoprosencephaly, neural tube defects suchas anencephaly which includes hydrangencephaly, Arnold-Chairi Deformity,encephalocele, meningocele, meningomyelocele, spinal dysraphism such asspina bifida cystica and spina bifida occulta.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include hereditary motor and sensory neuropathieswhich include Charcot-Marie Disease, Hereditary optic atrophy, Refsum'sDisease, hereditary spastic paraplegia, Werdnig-Hoffmann Disease,Hereditary Sensory and Autonomic Neuropathies such as CongenitalAnalgesia and Familial Dysautonomia, Neurologic manifestations (such asagnosia that include Gerstmann's Syndrome, Amnesia such as retrogradeamnesia, apraxia, neurogenic bladder, cataplexy, communicative disorderssuch as hearing disorders that includes deafness, partial hearing loss,loudness recruitment and tinnitus, language disorders such as aphasiawhich include agraphia, anomia, broca aphasia, and Wemicke Aphasia,Dyslexia such as Acquired Dyslexia, language development disorders,speech disorders such as aphasia which includes anomia, broca aphasiaand Wemicke Aphasia, articulation disorders, communicative disorderssuch as speech disorders which include dysarthria, echolalia, mutism andstuttering, voice disorders such as aphonia and hoarseness, decerebratestate, delirium, fasciculation, hallucinations, meningism, movementdisorders such as angelman syndrome, ataxia, athetosis, chorea,dystonia, hypokinesia, muscle hypotonia, myoclonus, tic, torticollis andtremor, muscle hypertonia such as muscle rigidity such as stiff-mansyndrome, muscle spasticity, paralysis such as facial paralysis whichincludes Herpes Zoster Oticus, Gastroparesis, Hemiplegia,ophthalmoplegia such as diplopia, Duane's Syndrome, Homer's Syndrome,Chronic progressive external ophthalmoplegia such as Keams Syndrome,Bulbar Paralysis, Tropical Spastic Paraparesis, Paraplegia such asBrown-Sequard Syndrome, quadriplegia, respiratory paralysis and vocalcord paralysis, paresis, phantom limb, taste disorders such as ageusiaand dysgeusia, vision disorders such as amblyopia, blindness, colorvision defects, diplopia, hemianopsia, scotoma and subnormal vision,sleep disorders such as hypersomnia which includes Kleine-LevinSyndrome, insomnia, and somnambulism, spasm such as trismus,unconsciousness such as coma, persistent vegetative state and syncopeand vertigo, neuromuscular diseases such as amyotonia congenita,amyotrophic lateral sclerosis, Lambert-Eaton Myasthenic Syndrome, motorneuron disease, muscular atrophy such as spinal muscular atrophy,Charcot-Marie Disease and Werdnig-Hoffmann Disease, PostpoliomyelitisSyndrome, Muscular Dystrophy, Myasthenia Gravis, Myotonia Atrophica,Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis,Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-ManSyndrome, peripheral nervous system diseases such as acrodynia, amyloidneuropathies, autonomic nervous system diseases such as Adie's Syndrome,Barre-Lieou Syndrome, Familial Dysautonomia, Homer's Syndrome, ReflexSympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseasessuch as Acoustic Nerve Diseases such as Acoustic Neuroma which includesNeurofibromatosis 2, Facial Nerve Diseases such as FacialNeuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders whichincludes amblyopia, nystagmnus, oculomotor nerve paralysis,ophthalmoplegia such as Duane's Syndrome, Homer's Syndrome, ChronicProgressive External Ophthalmoplegia which includes Kearns Syndrome,Strabismus such as Esotropia and Exotropia, Oculomotor Nerve Paralysis,Optic Nerve Diseases such as Optic Atrophy which includes HereditaryOptic Atrophy, Optic Disk Drusen, Optic Neuritis such as NeuromyelitisOptica, Papilledema, Trigeminal Neuralgia, Vocal Cord Paralysis,Demyelinating Diseases such as Neuromyelitis Optica and Swayback, andDiabetic neuropathies such as diabetic foot.

Additional neurologic diseases which can be treated or detected withpolynucleotides, polypeptides, agonists, and/or antagonists of thepresent invention include nerve compression syndromes such as carpaltunnel syndrome, tarsal tunnel syndrome, thoracic outlet syndrome suchas cervical rib syndrome, ulnar nerve compression syndrome, neuralgiasuch as causalgia, cervico-brachial neuralgia, facial neuralgia andtrigeminal neuralgia, neuritis such as experimental allergic neuritis,optic neuritis, polyneuritis, polyradiculoneuritis and radiculities suchas polyradiculitis, hereditary motor and sensory neuropathies such asCharcot-Marie Disease, Hereditary Optic Atrophy, Refsum's Disease,Hereditary Spastic Paraplegia and Werdnig-Hoffmann Disease, HereditarySensory and Autonomic Neuropathies which include Congenital Analgesiaand Familial Dysautonornia, POEMS Syndrome, Sciatica, Gustatory Sweatingand Tetany).

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing the polynucleotides or polypeptides,and/or agonists or antagonists of the invention, for therapeuticpurposes, for example, to stimulate epithelial cell proliferation andbasal keratinocytes for the purpose of wound healing, and to stimulatehair follicle production and healing of dermal wounds. Polynucleotidesor polypeptides, as well as agonists or antagonists of the invention,may be clinically useful in stimulating wound healing including surgicalwounds, excisional wounds, deep wounds involving damage of the dermisand epidermis, eye tissue wounds, dental tissue wounds, oral cavitywounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers,venous stasis ulcers, bums resulting from heat exposure or chemicals,and other abnormal wound healing conditions such as uremia,malnutrition, vitamin deficiencies and complications associted withsystemic treatment with steroids, radiation therapy and antineoplasticdrugs and antimetabolites. Polynucleotides or polypeptides, and/oragonists or antagonists of the invention, could be used to promotedermal reestablishment subsequent to dermal loss

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could be used to increase the adherence of skin grafts toa wound bed and to stimulate re-epithelialization from the wound bed.The following are a non-exhaustive list of grafts that polynucleotidesor polypeptides, agonists or antagonists of the invention, could be usedto increase adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepdermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, can be used to promote skin strength and to improve theappearance of aged skin.

It is believed that the polynucleotides or polypeptides, and/or agonistsor antagonists of the invention, will also produce changes in hepatocyteproliferation, and epithelial cell proliferation in the lung, breast,pancreas, stomach, small intesting, and large intestine. Thepolynucleotides or polypeptides, and/or agonists or antagonists of theinvention, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may promote proliferation of endothelialcells, keratinocytes, and basal keratinocytes.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could also be used to reduce the side effects of guttoxicity that result from radiation, chemotherapy treatments or viralinfections. The polynucleotides or polypeptides, and/or agonists orantagonists of the invention, may have a cytoprotective effect on thesmall intestine mucosa. The polynucleotides or polypeptides, and/oragonists or antagonists of the invention, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could further be used in full regeneration of skin infull and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment of other skin defects such as psoriasis. The polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to treat epidermolysis bullosa, a defect in adherence of theepidermis to the underlying dermis which results in frequent, open andpainful blisters by accelerating reepithelialization of these lesions.The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could also be used to treat gastric and doudenal ulcersand help heal by scar formation of the mucosal lining and regenerationof glandular mucosa and duodenal mucosal lining more rapidly.Inflammatory bowel diseases, such as Crohn's disease and ulcerativecolitis, are diseases which result in destruction of the mucosal surfaceof the small or large intestine, respectively. Thus, the polynucleotidesor polypeptides, and/or agonists or antagonists of the invention, couldbe used to promote the resurfacing of the mucosal surface to aid morerapid healing and to prevent progression of inflammatory bowel disease.Treatment with the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, is expected to have a significant effecton the production of mucus throughout the gastrointestinal tract andcould be used to protect the intestinal mucosa from injurious substancesthat are ingested or following surgery. The polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to treat diseases associate with the under expression of thepolynucleotides of the invention.

Moreover, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used to prevent and heal damageto the lungs due to various pathological states. A growth factor such asthe polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, which could stimulate proliferation and differentiationand promote the repair of alveoli and brochiolar epithelium to preventor treat acute or chronic lung damage. For example, emphysema, whichresults in the progressive loss of aveoli, and inhalation injuries,i.e., resulting from smoke inhalation and bums, that cause necrosis ofthe bronchiolar epithelium and alveoli could be effectively treatedusing the polynucleotides or polypeptides, and/or agonists orantagonists of the invention. Also, the polynucleotides or polypeptides,and/or agonists or antagonists of the invention, could be used tostimulate the proliferation of and differentiation of type IIpneumocytes, which may help treat or prevent disease such as hyalinemembrane diseases, such as infant respiratory distress syndrome andbronchopulmonary displasia, in premature infants.

The polynucleotides or polypeptides, and/or agonists or antagonists ofthe invention, could stimulate the proliferation and differentiation ofhepatocytes and, thus, could be used to alleviate or treat liverdiseases and pathologies such as fulminant liver failure caused bycirrhosis, liver damage caused by viral hepatitis and toxic substances(i.e., acetaminophen, carbon tetraholoride and other hepatotoxins knownin the art).

In addition, the polynucleotides or polypeptides, and/or agonists orantagonists of the invention, could be used treat or prevent the onsetof diabetes mellitus. In patients with newly diagnosed Types I and IIdiabetes, where some islet cell function remains, the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused to maintain the islet function so as to alleviate, delay or preventpermanent manifestation of the disease. Also, the polynucleotides orpolypeptides, and/or agonists or antagonists of the invention, could beused as an auxiliary in islet cell transplantation to improve or promoteislet cell function.

Infectious Disease

A polypeptide or polynucleotide and/or agonist or antagonist of thepresent invention can be used to treat or detect infectious agents. Forexample, by increasing the immune response, particularly increasing theproliferation and differentiation of B and/or T cells, infectiousdiseases may be treated. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, polypeptide or polynucleotide and/or agonist orantagonist of the present invention may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated or detected by a polynucleotide orpolypeptide and/or agonist or antagonist of the present invention.Examples of viruses, include, but are not limited to Examples ofviruses, include, but are not limited to the following DNA and RNAviruses and viral families: Arbovirus, Adenoviridae, Arenaviridae,Arterivirus, Bimaviridae, Bunyaviridae, Caliciviridae, Circoviridae,Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae(Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex,Herpes Zoster), Mononegavirus (e.g., Paramyxovindae, Morbillivirus,Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, andparainfluenza), Papiloma virus, Papovaviridae, Parvoviridae,Picomaviridae, Poxviridae (such as Smallpox or Vaccinia), Reoviridae(e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), andTogaviridae (e.g., Rubivirus). Viruses falling within these families cancause a variety of diseases or symptoms, including, but not limited to:arthritis, bronchiollitis, respiratory syncytial virus, encephalitis,eye infections (e.g., conjunctivitis, keratitis), chronic fatiguesyndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese Bencephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever,meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt'sLymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza,Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitteddiseases, skin diseases (e.g., Kaposi's, warts), and viremia.polynucleotides or polypeptides, or agonists or antagonists of theinvention, can be used to treat or detect any of these symptoms ordiseases. In specific embodiments, polynucleotides, polypeptides, oragonists or antagonists of the invention are used to treat: meningitis,Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additionalspecific embodiment polynucleotides, polypeptides, or agonists orantagonists of the invention are used to treat patients nonresponsive toone or more other commercially available hepatitis vaccines. In afurther specific embodiment polynucleotides, polypeptides, or agonistsor antagonists of the invention are used to treat AIDS.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated or detected by a polynucleotide or polypeptideand/or agonist or antagonist of the present invention include, but notlimited to, include, but not limited to, the following Gram-Negative andGram-positive bacteria and bacterial families and fungi: Actinomycetales(e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcusneofonnans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium),Bacteroidaceae, Blastomycosis, Bordetelia, Borrelia (e.g., Borreliaburgdorferi, Brucellosis, Candidiasis, Campylobacter,Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g.,Enterotoxigenic E. coli and Enterohemonrhagic E. coli),Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi, andSalmonella paratyphi), Serratia, Yersinia), Erysipelothrix,Helicobacter, Legionellosis, Leptospirosis, Listena, Mycoplasmatales,Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g.,Acinctobacter, Gonorrhea, Menigococcal), Meisseria meningitidis,Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g.,Heamophilus influenza type B), Pasteurella), Pseudomonas,Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal,Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcuspneumoniae and Group B Streptococcus). These bacterial or fungalfamilies can cause the following diseases or symptoms, including, butnot limited to: bacteremia, endocarditis, eye infections(conjunctivitis, tuberculosis, uveitis), gingivitis, opportunisticinfections (e.g., AIDS related infections), paronychia,prosthesis-related infections, Reiter's Disease, respiratory tractinfections, such as Whooping Cough or Empyema, sepsis, Lyme Disease,Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning,Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A andB), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. Polynucleotides or polypeptides, agonists orantagonists of the invention, can be used to treat or detect any ofthese symptoms or diseases. In specific embodiments, Ppolynucleotides,polypeptides, agonists or antagonists of the invention are used totreat: tetanus, Diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated or detected by a polynucleotide or polypeptide and/or agonist orantagonist of the present invention include, but not limited to, thefollowing families or class: Amebiasis, Babesiosis, Coccidiosis,Cryptosporidiosis, Dientarnoebiasis, Dourine, Ectoparasitic, Giardiasis,Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax,Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). Theseparasites can cause a variety of diseases or symptoms, including, butnot limited to: Scabies, Trombiculiasis, eye infections, intestinaldisease (e.g., dysentery, giardiasis), liver disease, lung disease,opportunistic infections (e.g., AIDS related), malaria, pregnancycomplications, and toxoplasmosis. polynucleotides or polypeptides, oragonists or antagonists of the invention, can be used to treat or detectany of these symptoms or diseases. In specific embodiments,polynucleotides, polypeptides, or agonists or antagonists of theinvention are used to treat malaria.

Preferably, treatment using a polypeptide or polynucleotide and/oragonist or antagonist of the present invention could-either be byadministering an effective amount of a polypeptide to the patient, or byremoving cells from the patient, supplying the cells with apolynucleotide of the present invention, and returning the engineeredcells to the patient (ex vivo therapy). Moreover, the polypeptide orpolynucleotide of the present invention can be used as an antigen in avaccine to raise an immune response against infectious disease.

Regeneration

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention can be used to differentiate, proliferate, and attractcells, leading to the regeneration of tissues. (See, Science 276:59-87(1997).) The regeneration of tissues could be used to repair, replace,or protect tissue damaged by congenital defects, trauma (wounds, bums,incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarhritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, a polynucleotide or polypeptide and/or agonist or antagonistof the present invention may increase regeneration of tissues difficultto heal. For example, increased tendon/ligament regeneration wouldquicken recovery time after damage. A polynucleotide or polypeptideand/or agonist or antagonist of the present invention could also be usedprophylactically in an effort to avoid damage. Specific diseases thatcould be treated include of tendinitis, carpal tunnel syndrome, andother tendon or ligament defects. A further example of tissueregeneration of non-healing wounds includes pressure ulcers, ulcersassociated with vascular insufficiency, surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by using apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention to proliferate and differentiate nerve cells. Diseasesthat could be treated using this method include central and peripheralnervous system diseases, neuropathies, or mechanical and traumaticdisorders (e.g., spinal cord disorders, head trauma, cerebrovasculardisease, and stoke). Specifically, diseases associated with peripheralnerve injuries, peripheral neuropathy (e.g., resulting from chemotherapyor other medical therapies), localized neuropathies, and central nervoussystem diseases (e.g., Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis, and Shy-Dragersyndrome), could all be treated using the polynucleotide or polypeptideand/or agonist or antagonist of the present invention.

Chemotaxis

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may have chemotaxis activity. A chemotaxic moleculeattracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils,T-cells, mast cells, eosinophils, epithelial and/or endothelial cells)to a particular site in the body, such as inflammation, infection, orsite of hyperproliferation. The mobilized cells can then fight offand/or heal the particular trauma or abnormality.

A polynucleotide or polypeptide and/or agonist or antagonist of thepresent invention may increase chemotaxic activity of particular cells.These chemotactic molecules can then be used to treat inflammation,infection, hyperproliferative disorders, or any immune system disorderby increasing the number of cells targeted to a particular location inthe body. For example, chemotaxic molecules can be used to treat woundsand other trauma to tissues by attracting immune cells to the injuredlocation. Chemotactic molecules of the present invention can alsoattract fibroblasts, which can be used to treat wounds.

It is also contemplated that a polynucleotide or polypeptide and/oragonist or antagonist of the present invention may inhibit chemotacticactivity. These molecules could also be used to treat disorders. Thus, apolynucleotide or polypeptide and/or agonist or antagonist of thepresent invention could be used as an inhibitor of chemotaxis.

Binding Activity

A polypeptide of the present invention may be used to screen formolecules that bind to the polypeptide or for molecules to which thepolypeptide binds. The binding of the polypeptide and the molecule mayactivate (agonist), increase, inhibit (antagonist), or decrease activityof the polypeptide or the molecule bound. Examples of such moleculesinclude antibodies, oligonucleotides, proteins (e.g., receptors), orsmall molecules.

Preferably, the molecule is closely related to the natural ligand of thepolypeptide, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which thepolypeptide binds, or at least, a fragment of the receptor capable ofbeing bound by the polypeptide (e.g. active site). In either case, themolecule can be rationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express the polypeptide, either as a secretedprotein or on the cell membrane. Preferred cells include cells frommammals, yeast, Drosophila, or E. coli. Cells expressing the polypeptide(or cell membrane containing the expressed polypeptide) are thenpreferably contacted with a test compound potentially containing themolecule to observe binding, stimulation, or inhibition of activity ofeither the polypeptide or the molecule.

The assay may simply test binding of a candidate compound to thepolypeptide, wherein binding is detected by a label, or in an assayinvolving competition with a labeled competitor. Further, the assay maytest whether the candidate compound results in a signal generated bybinding to the polypeptide.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining a polypeptide, measuring polypeptide/molecule activity orbinding, and comparing the polypeptide/molecule activity or binding to astandard.

Preferably, an ELISA assay can measure polypeptide level or activity ina sample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure polypeptide level or activity byeither binding, directly or indirectly, to the polypeptide or bycompeting with the polypeptide for a substrate.

Additionally, the receptor to which a polypeptide of the invention bindscan be identified by numerous methods known to those of skill in theart, for example, ligand panning and FACS sorting (Coligan, et al.,Current Protocols in Immun., 1(2), Chapter 5, (1991)). For example,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of polypeptidesof the invention thereby effectively generating agonists and antagonistsof polypeptides of the invention. See generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten,P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S.Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al., J. Mol.Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R. Biotechniques24(2):308-13 (1998)(each of these patents and publications are herebyincorporated by reference). In one embodiment, alteration ofpolynucleotides and corresponding polypeptides of the invention may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments into a desired polynucleotide sequence of theinvention molecule by homologous, or site-specific, recombination. Inanother embodiment, polynucleotides and corresponding polypeptides ofthe invention may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of the polypeptides of theinvention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules. In preferred embodiments, the heterologous molecules arefamily members. In further preferred embodiments, the heterologousmolecule is a growth factor such as, for example, platelet-derivedgrowth factor (PDGF), insulin-like growth factor (IGF-I), transforminggrowth factor (TGF)-alpha, epidermal growth factor (EGF), fibroblastgrowth factor (FGF), TGF-beta, bone morphogenetic protein (BMP)-2,BMP-4, BMP-5, BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A,OP-2, dorsalin, growth differentiation factors (GDFs), nodal, M1S,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active fragments of thepolypeptides of the invention. Biologically active fragments are thoseexhibiting activity similar, but not necessarily identical, to anactivity of the polypeptide. The biological activity of the fragmentsmay include an improved desired activity, or a decreased undesirableactivity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and 3[H] thyzmidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of 3[H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of 3[H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the receptor is measured and the ability of the compound tobind to the receptor and elicit a second messenger response is measuredto determine if the compound is a potential agonist or antagonist. Suchsecond messenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat disease or to bring about a particular result in a patient (e.g.,blood vessel growth) by activating or inhibiting thepolypeptide/molecule. Moreover, the assays can discover agents which mayinhibit or enhance the production of the polypeptides of the inventionfrom suitably manipulated cells or tissues. Therefore, the inventionincludes a method of identifying compounds which bind to thepolypeptides of the invention comprising the steps of: (a) incubating acandidate binding compound with the polypeptide; and (b) determining ifbinding has occurred. Moreover, the invention includes a method ofidentifying agonists/antagonists comprising the steps of (a) incubatinga candidate compound with the polypeptide, (b) assaying a biologicalactivity, and (b) determining if a biological activity of thepolypeptide has been altered.

Also, one could identify molecules bind a polypeptide of the inventionexperimentally by using the beta-pleated sheet regions contained in thepolypeptide sequence of the protein. Accordingly, specific embodimentsof the invention are directed to polynucleotides encoding polypeptideswhich comprise, or alternatively consist of, the amino acid sequence ofeach beta pleated sheet regions in a disclosed polypeptide sequence.Additional embodiments of the invention are directed to polynucleotidesencoding polypeptides which comprise, or alternatively consist of, anycombination or all of contained in the polypeptide sequences of theinvention. Additional preferred embodiments of the invention aredirected to polypeptides which comprise, or alternatively consist of,the amino acid sequence of each of the beta pleated sheet regions in oneof the polypeptide sequences of the invention. Additional embodiments ofthe invention are directed to polypeptides which comprise, oralternatively consist of any combination or all of the beta pleatedsheet regions in one of the polypeptide sequences of the invention.

Targeted Delivery

In another embodiment, the invention provides a method of deliveringcompositions to targeted cells expressing a receptor for a polypeptideof the invention, or cells expressing a cell bound form of a polypeptideof the invention.

As discussed herein, polypeptides or antibodies of the invention may beassociated with heterologous polypeptides, heterologous nucleic acids,toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalentinteractions. In one embodiment, the invention provides a method for thespecific delivery of compositions of the invention to cells byadministering polypeptides of the invention (including antibodies) thatare associated with heterologous polypeptides or nucleic acids. In oneexample, the invention provides a method for delivering a therapeuticprotein into the targeted cell. In another example, the inventionprovides a method for delivering a single stranded nucleic acid (e.g.,antisense or ribozymes) or double stranded nucleic acid (e.g., DNA thatcan integrate into the cell's genome or replicate episomally and thatcan be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., polypeptides of theinvention or antibodies of the invention) in association with toxins orcytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, or any molecules or enzymes not normallypresent in or on the surface of a cell that under defined conditionscause the cell's death. Toxins that may be used according to the methodsof the invention include, but are not limited to, radioisotopes known inthe art, compounds such as, for example, antibodies (or complementfixing containing portions thereof) that bind an inherent or inducedendogenous cytotoxic effector system, thymidine kinase, endonuclease,RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheriatoxin, saporin, momordin, gelonin, pokeweed antiviral protein,alpha-sarcin and cholera toxin. By “cytotoxic prodrug” is meant anon-toxic compound that is converted by an enzyme, normally present inthe cell, into a cytotoxic compound. Cytotoxic prodrugs that may be usedaccording to the methods of the invention include, but are not limitedto, glutamyl derivatives of benzoic acid mustard alkylating agent,phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside,daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

Drug Screening

Further contemplated is the use of the polypeptides of the presentinvention, or the polynucleotides encoding these polypeptides, to screenfor molecules which modify the activities of the polypeptides of thepresent invention. Such a method would include contacting thepolypeptide of the present invention with a selected compound(s)suspected of having antagonist or agonist activity, and assaying theactivity of these polypeptides following binding.

This invention is particularly useful for screening therapeuticcompounds by using the polypeptides of the present invention, or bindingfragments thereof, in any of a variety of drug screening techniques. Thepolypeptide or fragment employed in such a test may be affixed to asolid support, expressed on a cell surface, free in solution, or locatedintracellularly. One method of drug screening utilizes eukaryotic orprokaryotic host cells which are stably transformed with recombinantnucleic acids expressing the polypeptide or fragment. Drugs are screenedagainst such transformed cells in competitive binding assays. One maymeasure, for example, the formulation of complexes between the agentbeing tested and a polypeptide of the present invention.

Thus, the present invention provides methods of screening for drugs orany other agents which affect activities mediated by the polypeptides ofthe present invention. These methods comprise contacting such an agentwith a polypeptide of the present invention or a fragment thereof andassaying for the presence of a complex between the agent and thepolypeptide or a fragment thereof, by methods well known in the art. Insuch a competitive binding assay, the agents to screen are typicallylabeled. Following incubation, free agent is separated from that presentin bound form, and the amount of free or uncomplexed label is a measureof the ability of a particular agent to bind to the polypeptides of thepresent invention.

Another technique for drug screening provides high throughput screeningfor compounds having suitable binding affinity to the polypeptides ofthe present invention, and is described in great detail in EuropeanPatent Application 84/03564, published on Sep. 13, 1984, which isincorporated herein by reference herein. Briefly stated, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, such as plastic pins or some other surface. The peptide testcompounds are reacted with polypeptides of the present invention andwashed. Bound polypeptides are then detected by methods well known inthe art. Purified polypeptides are coated directly onto plates for usein the aforementioned drug screening techniques. In addition,non-neutralizing antibodies may be used to capture the peptide andimmobilize it on the solid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralizing antibodies capable of binding polypeptidesof the present invention specifically compete with a test compound forbinding to the polypeptides or fragments thereof. In this manner, theantibodies are used to detect the presence of any peptide which sharesone or more antigenic epitopes with a polypeptide of the invention.

Antisense And Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:X, or the complementary strand thereof, and/or to nucleotidesequences contained a deposited clone. In one embodiment, antisensesequence is generated internally by the organism, in another embodiment,the antisense sequence is separately administered (see, for example,O'Connor, Neurochem., 56:560 (1991). Oligodeoxynucleotides as AnitsenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988).Antisense technology can be used to control gene expression throughantisense DNA or RNA, or through triple-helix formation. Antisensetechniques are discussed for example, in Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research,6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan etal., Science, 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the use of c-myc and c-myb antisense RNA constructs toinhibit the growth of the non-lymphocytic leukemia cell line HL-60 andother cell lines was previously described. (Wickstrom et al. (1988);Anfossi et al. (1989)). These experiments were performed in vitro byincubating cells with the oligoribonucleotide. A similar procedure forin vivo use is described in WO 91/15580. Briefly, a pair ofoligonucleotides for a given antisense RNA is produced as follows: Asequence complimentary to the first 15 bases of the open reading frameis flanked by an EcoR1 site on the 5 end and a HindIII site on the 3end. Next, the pair of oligonucleotides is heated at 90° C. for oneminute and then annealed in 2× ligation buffer (20 mM TRIS HCl pH 7.5,10 mM MgCl2, 10 MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligatedto the EcoRI/Hind III site of the retroviral vector PMV7 (WO 91/15580).

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the antisense nucleic acid of the invention.Such a vector can remain episomal or become chromosomally integrated, aslong as it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others known inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding a polypeptide of the invention, orfragments thereof, can be by any promoter known in the art to act invertebrate, preferably human cells. Such promoters can be inducible orconstitutive. Such promoters include, but are not limited to, the SV40early promoter region (Bemoist and Chambon, Nature, 29:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell, 22:787-797 (1980), the herpes thymidinepromoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A., 78:1441-1445(1981), the regulatory sequences of the metallothionein gene (Brinsteret al., Nature, 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a portion of an RNA transcript of a gene ofinterest. However, absolute complementarity, although preferred, is notrequired. A sequence “complementary to at least a portion of an RNA,”referred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double stranded antisense nucleic acids of the invention, asingle strand of the duplex DNA may thus be tested, or triplex formationmay be assayed. The ability to hybridize will depend on both the degreeof complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a RNA sequence of the invention it may contain and stillform a stable duplex (or triplex as the case may be). One skilled in theart can ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., Nature,372:333-335 (1994). Thus, oligonucleotides complementary to either the5′-or 3′-non-translated, non-coding regions of a polynucleotide sequenceof the invention could be used in an antisense approach to inhibittranslation of endogenous mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′-or coding region of mRNA, antisense nucleic acids should be atleast six nucleotides in length, and are preferably oligonucleotidesranging from 6 to about 50 nucleotides in length. In specific aspectsthe oligonucleotide is at least 10 nucleotides, at least 17 nucleotides,at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A.86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci., 84:648-652(1987); PCT Publication NO: WO88/09810, published Dec. 15, 1988) or theblood-brain barrier (see, e.g., PCT Publication No: WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., BioTechniques, 6:958-976 (1988)) or intercalatingagents. (See, e.g., Zon, Pharm. Res., 5:539-549 (1988)). To this end,the oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcyosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic and methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids Res., 15:6625-6641 (1987)). The oligonucleotide is a2-0-methylribonucleotide (Inoue et al., Nucl. Acids Res., 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res., 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci.U.S.A., 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to the coding region sequenceof the invention could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science, 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy mRNAs corresponding to thepolynucleotides of the invention, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art and is described morefully in Haseloff and Gerlach, Nature, 334:585-591 (1988). There arenumerous potential hammerhead ribozyme cleavage sites within eachnucleotide sequence disclosed in the sequence listing. Preferably, theribozyme is engineered so that the cleavage recognition site is locatednear the 5′ end of the mRNA corresponding to the polynucleotides of theinvention; i.e., to increase efficiency and minimize the intracellularaccumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express thepolynucleotides of the invention in vivo. DNA constructs encoding theribozyme may be introduced into the cell in the same manner as describedabove for the introduction of antisense encoding DNA. A preferred methodof delivery involves using a DNA construct “encoding” the ribozyme underthe control of a strong constitutive promoter, such as, for example, polIII or pol II promoter, so that transfected cells will producesufficient quantities of the ribozyme to destroy endogenous messages andinhibit translation. Since ribozymes unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat the diseasesdescribed herein.

Thus, the invention provides a method of treating disorders or diseases,including but not limited to the disorders or diseases listed throughoutthis application, associated with overexpression of a polynucleotide ofthe present invention by administering to a patient (a) an antisensemolecule directed to the polynucleotide of the present invention, and/or(b) a ribozyme directed to the polynucleotide of the present invention.

Binding Peptides and Other Molecules

The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind secreted polypeptides, andthe secreted binding molecules identified thereby. These bindingmolecules are useful, for example, as agonists and antagonists of thesecreted polypeptides. Such agonists and antagonists can be used, inaccordance with the invention, in the therapeutic embodiments describedin detail, below.

This method comprises the steps of:

a. contacting secreted polypeptides or secreted-like polypeptides with aplurality of molecules; and

b. identifying a molecule that binds the secreted polypeptides orsecreted-like polypeptides.

The step of contacting the secreted polypeptides or secreted-likepolypeptides with the plurality of molecules may be effected in a numberof ways. For example, one may contemplate immobilizing the secretedpolypeptides or secreted-like polypeptides on a solid support andbringing a solution of the plurality of molecules in contact with theimmobilized secreted polypeptides or secreted-like polypeptides. Such aprocedure would be akin to an affinity chromatographic process, with theaffinity matrix being comprised of the immobilized secreted polypeptidesor secreted-like polypeptides. The molecules having a selective affinityfor the secreted polypeptides or secreted-like polypeptides can then bepurified by affinity selection. The nature of the solid support, processfor attachment of the secreted polypeptides or secreted-likepolypeptides to the solid support, solvent, and conditions of theaffinity isolation or selection are largely conventional and well knownto those of ordinary skill in the art.

Alternatively, one may also separate a plurality of polypeptides intosubstantially separate fractions comprising a subset of or individualpolypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” by the secreted polypeptides or secreted-like polypeptides,optionally in the presence of an inducer should one be required forexpression, to determine if any selective affinity interaction takesplace between the secreted polypeptides or secreted-like polypeptidesand the individual clone. Prior to contacting the secreted polypeptidesor secreted-like polypeptides with each fraction comprising individualpolypeptides, the polypeptides could first be transferred to a solidsupport for additional convenience. Such a solid support may simply be apiece of filter membrane, such as one made of nitrocellulose or nylon.In this manner, positive clones could be identified from a collection oftransformed host cells of an expression library, which harbor a DNAconstruct encoding a polypeptide having a selective affinity forsecreted polypeptides or secreted-like polypeptides. Furthermore, theamino acid sequence of the polypeptide having a selective affinity forthe secreted polypeptides or secreted-like polypeptides can bedetermined directly by conventional means or the coding sequence of theDNA encoding the polypeptide can frequently be determined moreconveniently. The primary sequence can then be deduced from thecorresponding DNA sequence. If the amino acid sequence is to bedetermined from the polypeptide itself, one may use microsequencingtechniques. The sequencing technique may include mass spectroscopy.

In certain situations, it may be desirable to wash away any unboundsecreted polypeptides or secreted-like polypeptides, or alternatively,unbound polypeptides, from a mixture of the secreted polypeptides orsecreted-like polypeptides and the plurality of polypeptides prior toattempting to determine or to detect the presence of a selectiveaffinity interaction. Such a wash step may be particularly desirablewhen the secreted polypeptides or secreted-like polypeptides or theplurality of polypeptides is bound to a solid support.

The plurality of molecules provided according to this method may beprovided by way of diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries which can be screened for molecules thatspecifically bind secreted polypeptides. Many libraries are known in theart that can be used, e.g., chemically synthesized libraries,recombinant (e.g., phage display libraries), and in vitrotranslation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., 1991, Science 251:767-773;Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature354:82-84; Medynski, 1994, Bio/Technology 12:709-710;Gallop et al.,1994, J. Medicinal Chemistry 37(9):1233-1251; Ohimeyer et al, 1993,Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl.Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA91:1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lemer,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith,1990, Science 249:386-390; Devlin et al. 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

The variety of non-peptide libraries that are useful in the presentinvention is great. For example, Ecker and Crooke, 1995, Bio/Technology13:351-360 list benzodiazepines, hydantoins, piperazinediones,biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids,acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, andoxazolones as among the chemical species that form the basis of variouslibraries.

Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

Non-peptide oligomer libraries utilize a large number of monomers thatare assembled together in ways that create new shapes that depend on theorder of the monomers. Among the monomer units that have been used arecarbamates, pyrrolinones, and morpholinos. Peptoids, peptide-likeoligomers in which the side chain is attached to the alpha amino grouprather than the alpha carbon, form the basis of another version ofnon-peptide oligomer libraries. The first non-peptide oligomer librariesutilized a single type of monomer and thus contained a repeatingbackbone. Recent libraries have utilized more than one monomer, givingthe libraries added flexibility.

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. Nos.5,096,815, 5,223,409, and 5,198,346, all to Ladner et al.; Rebar andPabo, 1993, Science 263:671-673; and CT Publication No. WO 94/18318.

In a specific embodiment, screening to identify a molecule that bindssecreted polypeptides can be carried out by contacting the librarymembers with a secreted polypeptides or secreted-like polypeptidesimmobilized on a solid phase and harvesting those library members thatbind to the secreted polypeptides or secreted-like polypeptides.Examples of such screening methods, termed “panning” techniques aredescribed by way of example in Parmley and Smith, 1988, Gene 73:305-318;Fowlkes et al., 1992, BioTechniques 13:422-427; PCT Publication No. WO94/18318; and in references cited herein.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien etal., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to secreted polypeptides orsecreted-like polypeptides.

Where the secreted binding molecule is a polypeptide, the polypeptidecan be conveniently selected from any peptide library, including randompeptide libraries, combinatorial peptide libraries, or biased peptidelibraries. The term “biased” is used herein to mean that the method ofgenerating the library is manipulated so as to restrict one or moreparameters that govern the diversity of the resulting collection ofmolecules, in this case peptides.

Thus, a truly random peptide library would generate a collection ofpeptides in which the probability of finding a particular amino acid ata given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid or that positions 4,8, and 9 of a decapeptide library be fixed to include only arginine.Clearly, many types of biases can be contemplated, and the presentinvention is not restricted to any particular bias. Furthermore, thepresent invention contemplates specific types of peptide libraries, suchas phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

As mentioned above, in the case of a secreted binding molecule that is apolypeptide, the polypeptide may have about 6 to less than about 60amino acid residues, preferably about 6 to about 10 amino acid residues,and most preferably, about 6 to about 22 amino acids. In anotherembodiment, a secreted binding polypeptide has in the range of 15-100amino acids, or 20-50 amino acids.

The selected secreted binding polypeptide can be obtained by chemicalsynthesis or recombinant expression.

Other Activities

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat and prevent neuronal damage which occurs incertain neuronal disorders or neuro-degenerative conditions such asAlzheimer's disease, Parkinson's disease, and AIDS-related complex. Thepolypeptide of the invention may have the ability to stimulatechondrocyte growth, therefore, they may be employed to enhance bone andperiodontal regeneration and aid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The polypeptide of the invention may also be employed for preventinghair loss, since FGF family members activate hair-forming cells andpromotes melanocyte growth. Along the same lines, the polypeptides ofthe present invention may be employed to stimulate growth anddifferentiation of hematopoietic cells and bone marrow cells when usedin combination with other cytokines.

The polypeptide of the invention may also be employed to maintain organsbefore transplantation or for supporting cell culture of primarytissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

The polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used to modulate mammaliancharacteristics, such as body height, weight, hair color, eye color,skin, percentage of adipose tissue, pigmentation, size, and shape (e.g.,cosmetic surgery). Similarly, polypeptides or polynucleotides and/oragonist or antagonists of the present invention may be used to modulatemammalian metabolism affecting catabolism, anabolism, processing,utilization, and storage of energy.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may be used to change a mammal's mental state orphysical state by influencing biorhythms, caricadic rhythms, depression(including depressive disorders), tendency for violence, tolerance forpain, reproductive capabilities (preferably by Activin or Inhibin-likeactivity), hormonal or endocrine levels, appetite, libido, memory,stress, or other cognitive qualities.

Polypeptide or polynucleotides and/or agonist or antagonists of thepresent invention may also be used as a food additive or preservative,such as to increase or decrease storage capabilities, fat content,lipid, protein, carbohydrate, vitamins, minerals, cofactors or othernutritional components.

Other Preferred Embodiments

Other preferred embodiments of the claimed invention include an isolatednucleic acid molecule comprising a nucleotide sequence which is at least95% identical to a sequence of at least about 50 contiguous nucleotidesin the nucleotide sequence of SEQ ID NO:X wherein X is any integer asdefined in Table XIV.

Also preferred is a nucleic acid molecule wherein said sequence ofcontiguous nucleotides is included in the nucleotide sequence of SEQ IDNO:X in the range of positions beginning with the nucleotide at aboutthe position of the 5′ Nucleotide of the Clone Sequence and ending withthe nucleotide at about the position of the 3′ Nucleotide of the CloneSequence as defined for SEQ ID NO:X in Table XIV.

Also preferred is a nucleic acid molecule wherein said sequence ofcontiguous nucleotides is included in the nucleotide sequence of SEQ IDNO:X in the range of positions beginning with the nucleotide at aboutthe position of the 5′ Nucleotide of the Start Codon and ending with thenucleotide at about the position of the 3′ Nucleotide of the CloneSequence as defined for SEQ ID NO:X in Table XIV.

Similarly preferred is a nucleic acid molecule wherein said sequence ofcontiguous nucleotides is included in the nucleotide sequence of SEQ IDNO:X in the range of positions beginning with the nucleotide at aboutthe position of the 5′ Nucleotide of the First Amino Acid of the SignalPeptide and ending with the nucleotide at about the position of the 3′Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in TableXIV.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 150 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

Further preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast about 500 contiguous nucleotides in the nucleotide sequence of SEQID NO:X.

A further preferred embodiment is a nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to the nucleotidesequence of SEQ ID NO:X beginning with the nucleotide at about theposition of the 5′ Nucleotide of the First Amino Acid of the SignalPeptide and ending with the nucleotide at about the position of the 3′Nucleotide of the Clone Sequence as defined for SEQ ID NO:X in TableXIV.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thecomplete nucleotide sequence of SEQ ID NO:X.

Also preferred is an isolated nucleic acid molecule which hybridizesunder stringent hybridization conditions to a nucleic acid molecule,wherein said nucleic acid molecule which hybridizes does not hybridizeunder stringent hybridization conditions to a nucleic acid moleculehaving a nucleotide sequence consisting of only A residues or of only Tresidues.

Also preferred is a composition of matter comprising a DNA moleculewhich comprises a human cDNA clone identified by a cDNA Clone Identifierin Table XIV, which DNA molecule is contained in the material depositedwith the American Type Culture Collection and given the ATCC DepositNumber shown in Table XIV for said cDNA Clone Identifier.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in the nucleotide sequence of a humancDNA clone identified by a cDNA Clone Identifier in Table XIV, which DNAmolecule is contained in the deposit given the ATCC Deposit Number shownin Table XIV.

Also preferred is an isolated nucleic acid molecule, wherein saidsequence of at least 50 contiguous nucleotides is included in thenucleotide sequence of the complete open reading frame sequence encodedby said human cDNA clone.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to sequence of atleast 150 contiguous nucleotides in the nucleotide sequence encoded bysaid human cDNA clone.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical tosequence of at least 500 contiguous nucleotides in the nucleotidesequence encoded by said human cDNA clone.

A further preferred embodiment is an isolated nucleic acid moleculecomprising a nucleotide sequence which is at least 95% identical to thecomplete nucleotide sequence encoded by said human cDNA clone.

A further preferred embodiment is a method for detecting in a biologicalsample a nucleic acid molecule comprising a nucleotide sequence which isat least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:X wherein X is any integer as definedin Table XIV; and a nucleotide sequence encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table XIV and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableXIV; which method comprises a step of comparing a nucleotide sequence ofat least one nucleic acid molecule in said sample with a sequenceselected from said group and determining whether the sequence of saidnucleic acid molecule in said sample is at least 95% identical to saidselected sequence.

Also preferred is the above method wherein said step of comparingsequences comprises determining the extent of nucleic acid hybridizationbetween nucleic acid molecules in said sample and a nucleic acidmolecule comprising said sequence selected from said group. Similarly,also preferred is the above method wherein said step of comparingsequences is performed by comparing the nucleotide sequence determinedfrom a nucleic acid molecule in said sample with said sequence selectedfrom said group. The nucleic acid molecules can comprise DNA moleculesor RNA molecules.

A further preferred embodiment is a method for identifying the species,tissue or cell type of a biological sample which method comprises a stepof detecting nucleic acid molecules in said sample, if any, comprising anucleotide sequence that is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from the groupconsisting of: a nucleotide sequence of SEQ ID NO:X wherein X is anyinteger as defined in Table XIV; and a nucleotide sequence encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table XIV andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table XIV.

The method for identifying the species, tissue or cell type of abiological sample can comprise a step of detecting nucleic acidmolecules comprising a nucleotide sequence in a panel of at least twonucleotide sequences, wherein at least one sequence in said panel is atleast 95% identical to a sequence of at least 50 contiguous nucleotidesin a sequence selected from said group.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a geneencoding a secreted protein identified in Table XIV, which methodcomprises a step of detecting in a biological sample obtained from saidsubject nucleic acid molecules, if any, comprising a nucleotide sequencethat is at least 95% identical to a sequence of at least 50 contiguousnucleotides in a sequence selected from the group consisting of: anucleotide sequence of SEQ ID NO:X wherein X is any integer as definedin Table XIV; and a nucleotide sequence encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table XIV and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableXIV.

The method for diagnosing a pathological condition can comprise a stepof detecting nucleic acid molecules comprising a nucleotide sequence ina panel of at least two nucleotide sequences, wherein at least onesequence in said panel is at least 95% identical to a sequence of atleast 50 contiguous nucleotides in a sequence selected from said group.

Also preferred is a composition of matter comprising isolated nucleicacid molecules wherein the nucleotide sequences of said nucleic acidmolecules comprise a panel of at least two nucleotide sequences, whereinat least one sequence in said panel is at least 95% identical to asequence of at least 50 contiguous nucleotides in a sequence selectedfrom the group consisting of: a nucleotide sequence of SEQ ID NO:Xwherein X is any integer as defined in Table XIV; and a nucleotidesequence encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table XIV and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table XIV. The nucleic acidmolecules can comprise DNA molecules or RNA molecules.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the amino acid sequence of SEQ ID. NO:Ywherein Y is any integer as defined in Table XIV.

Also preferred is a polypeptide, wherein said sequence of contiguousamino acids is included in the amino acid sequence of SEQ ID NO:Y in therange of positions beginning with the residue at about the position ofthe First Amino Acid of the Secreted Portion and ending with the residueat about the Last Amino Acid of the Open Reading Frame as set forth forSEQ ID NO:Y in Table XIV.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of SEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the complete amino acid sequence ofSEQ ID NO:Y.

Further preferred is an isolated polypeptide comprising an amino acidsequence at least 90% identical to a sequence of at least about 10contiguous amino acids in the complete amino acid sequence of a secretedprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table XIV and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table XIV.

Also preferred is a polypeptide wherein said sequence of contiguousamino acids is included in the amino acid sequence of a secreted portionof the secreted protein encoded by a human cDNA clone identified by acDNA Clone Identifier in Table XIV and contained in the deposit with theATCC Deposit Number shown for said cDNA clone in Table XIV.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 30contiguous amino acids in the amino acid sequence of the secretedportion of the protein encoded by a human cDNA clone identified by acDNA Clone Identifier in Table XIV and contained in the deposit with theATCC Deposit Number shown for said cDNA clone in Table XIV.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to a sequence of at least about 100contiguous amino acids in the amino acid sequence of the secretedportion of the protein encoded by a human cDNA clone identified by acDNA Clone Identifier in Table XIV and contained in the deposit with theATCC Deposit Number shown for said cDNA clone in Table XIV.

Also preferred is an isolated polypeptide comprising an amino acidsequence at least 95% identical to the amino acid sequence of thesecreted portion of the protein encoded by a human cDNA clone identifiedby a cDNA Clone Identifier in Table XIV and contained in the depositwith the ATCC Deposit Number shown for said cDNA clone in Table XIV.

Further preferred is an isolated antibody which binds specifically to apolypeptide comprising an amino acid sequence that is at least 90%identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y wherein Y is any integer as defined in Table XIV; and acomplete amino acid sequence of a protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table XIV and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableXIV.

Further preferred is a method for detecting in a biological sample apolypeptide comprising an amino acid sequence which is at least 90%identical to a sequence of at least 10 contiguous amino acids in asequence selected from the group consisting of an amino acid sequence ofSEQ ID NO:Y wherein Y is any integer as defined in Table XIV; and acomplete amino acid sequence of a protein encoded by a human cDNA cloneidentified by a cDNA Clone Identifier in Table XIV and contained in thedeposit with the ATCC Deposit Number shown for said cDNA clone in TableXIV; which method comprises a step of comparing an amino acid sequenceof at least one polypeptide molecule in said sample with a sequenceselected from said group and determining whether the sequence of saidpolypeptide molecule in said sample is at least 90% identical to saidsequence of at least 10 contiguous amino acids.

Also preferred is the above method wherein said step of comparing anamino acid sequence of at least one polypeptide molecule in said samplewith a sequence selected from said group comprises determining theextent of specific binding of polypeptides in said sample to an antibodywhich binds specifically to a polypeptide comprising an amino acidsequence that is at least 90% identical to a sequence of at least 10contiguous amino acids in a sequence selected from the group consistingof: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer asdefined in Table XIV; and a complete amino acid sequence of a proteinencoded by a human cDNA clone identified by a cDNA Clone Identifier inTable XIV and contained in the deposit with the ATCC Deposit Numbershown for said cDNA clone in Table XIV.

Also preferred is the above method wherein said step of comparingsequences is performed by comparing the amino acid sequence determinedfrom a polypeptide molecule in said sample with said sequence selectedfrom said group.

Also preferred is a method for identifying the species, tissue or celltype of a biological sample which method comprises a step of detectingpolypeptide molecules in said sample, if any, comprising an amino acidsequence that is at least 90% identical to a sequence of at least 10contiguous amino acids in a sequence selected from the group consistingof: an amino acid sequence of SEQ ID NO:Y wherein Y is any integer asdefined in Table XIV; and a complete amino acid sequence of a secretedprotein encoded by a human cDNA clone identified by a cDNA CloneIdentifier in Table XIV and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table XIV.

Also preferred is the above method for identifying the species, tissueor cell type of a biological sample, which method comprises a step ofdetecting polypeptide molecules comprising an amino acid sequence in apanel of at least two amino acid sequences, wherein at least onesequence in said panel is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the abovegroup.

Also preferred is a method for diagnosing in a subject a pathologicalcondition associated with abnormal structure or expression of a geneencoding a secreted protein identified in Table XIV, which methodcomprises a step of detecting in a biological sample obtained from saidsubject polypeptide molecules comprising an amino acid sequence in apanel of at least two amino acid sequences, wherein at least onesequence in said panel is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table XIV; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table XIV and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table XIV.

In any of these methods, the step of detecting said polypeptidemolecules includes using an antibody.

Also preferred is an isolated nucleic acid molecule comprising anucleotide sequence which is at least 95% identical to a nucleotidesequence encoding a polypeptide wherein said polypeptide comprises anamino acid sequence that is at least 90% identical to a sequence of atleast 10 contiguous amino acids in a sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table XIV; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table XIV and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table XIV.

Also preferred is an isolated nucleic acid molecule, wherein saidnucleotide sequence encoding a polypeptide has been optimized forexpression of said polypeptide in a prokaryotic host.

Also preferred is an isolated nucleic acid molecule, wherein saidpolypeptide comprises an amino acid sequence selected from the groupconsisting of: an amino acid sequence of SEQ ID NO:Y wherein Y is anyinteger as defined in Table XIV; and a complete amino acid sequence of asecreted protein encoded by a human cDNA clone identified by a cDNAClone Identifier in Table XIV and contained in the deposit with the ATCCDeposit Number shown for said cDNA clone in Table XIV.

Further preferred is a method of making a recombinant vector comprisinginserting any of the above isolated nucleic acid molecule into a vector.Also preferred is the recombinant vector produced by this method. Alsopreferred is a method of making a recombinant host cell comprisingintroducing the vector into a host cell, as well as the recombinant hostcell produced by this method.

Also preferred is a method of making an isolated polypeptide comprisingculturing this recombinant host cell under conditions such that saidpolypeptide is expressed and recovering said polypeptide. Also preferredis this method of making an isolated polypeptide, wherein saidrecombinant host cell is a eukaryotic cell and said polypeptide is asecreted portion of a human secreted protein comprising an amino acidsequence selected from the group consisting of: an amino acid sequenceof SEQ ID NO:Y beginning with the residue at the position of the FirstAmino Acid of the Secreted Portion of SEQ ID NO:Y wherein Y is aninteger set forth in Table XIV and said position of the First Amino Acidof the Secreted Portion of SEQ ID NO:Y is defined in Table XIV; and anamino acid sequence of a secreted portion of a protein encoded by ahuman cDNA clone identified by a cDNA Clone Identifier in Table XIV andcontained in the deposit with the ATCC Deposit Number shown for saidcDNA clone in Table XIV. The isolated polypeptide produced by thismethod is also preferred.

Also preferred is a method of treatment of an individual in need of anincreased level of a secreted protein activity, which method comprisesadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated polypeptide, polynucleotide, orantibody of the claimed invention effective to increase the level ofsaid protein activity in said individual.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Isolation of a Selected cDNA Clone From the DepositedSample

Each cDNA clone in a cited ATCC deposit is contained in a plasmidvector. Table XIV identifies the vectors used to construct the cDNAlibrary from which each clone was isolated. In many cases, the vectorused to construct the library is a phage vector from which a plasmid hasbeen excised. The table immediately below correlates the related plasmidfor each phage vector used in constructing the cDNA library. Forexample, where a particular clone is identified in Table XIV as beingisolated in the vector “Lambda Zap,” the corresponding deposited cloneis in “pBluescript.”

Vector Used to Construct Library Corresponding Deposited Plasmid LambdaZap pBluescript (pBS) Uni-Zap XR pBluescript (pBS) Zap Express pBKlafmid BA plafmid BA pSport1 pSport1 pCMVSport 2.0 pCMVSport 2.0pCMVSport 3.0 pCMVSport 3.0 pCR ® 2.1 pCR ® 2.1

Vectors Lambda Zap (U.S. Pat. Nos. 5,128,256 and 5,286,636), Uni-Zap XR(U.S. Pat. Nos. 5,128,256 and 5,286,636), Zap Express (U.S. Pat. Nos.5,128,256 and 5,286,636), pBluescript (pBS) (Short, J. M. et al.,Nucleic Acids Res. 16:7583-7600 (1988); Alting-Mees, M. A. and Short, J.M., Nucleic Acids Res. 17:9494 (1989)) and pBK (Alting-Mees, M. A. etal., Strategies 5:58-61 (1992)) are commercially available fromStratagene Cloning Systems, Inc., 11011 N. Torrey Pines Road, La Jolla,Calif., 92037. pBS contains an ampicillin resistance gene and pBKcontains a neomycin resistance gene. Both can be transformed into E.coli strain XL-1 Blue, also available from Stratagene. pBS comes in 4forms SK+, SK−, KS+ and KS. The S and K refers to the orientation of thepolylinker to the T7 and T3 primer sequences which flank the polylinkerregion (“S” is for SacI and “K” is for KpnI which are the first sites oneach respective end of the linker). “+” or “−” refer to the orientationof the f1 origin of replication (“ori”), such that in one orientation,single stranded rescue initiated from the f1 ori generates sense strandDNA and in the other, antisense.

Vectors pSport1, pCMVSport 2.0 and pCMVSport 3.0, were obtained fromLife Technologies, Inc., P.O. Box 6009, Gaithersburg, Md. 20897. AllSport vectors contain an ampicillin resistance gene and may betransformed into E. coli strain DH10B, also available from LifeTechnologies. (See, for instance, Gruber, C. E., et al., Focus 15:59(1993).) Vector lafmid BA (Bento Soares, Columbia University, NY)contains an ampicillin resistance gene and can be transformed into E.coli strain XL-1 Blue. Vector pCR®2.1, which is available fromInvitrogen, 1600 Faraday Avenue, Carlsbad, Calif. 92008, contains anampicillin resistance gene and may be transformed into E. coli strainDH10B, available from Life Technologies. (See, for instance, Clark, J.M., Nuc. Acids Res. 16:9677-9686 (1988) and Mead, D. et al.,Bio/Technology 9: (1991).) Preferably, a polynucleotide of the presentinvention does not comprise the phage vector sequences identified forthe particular clone in Table XIV, as well as the corresponding plasmidvector sequences designated above.

The deposited material in the sample assigned the ATCC Deposit Numbercited in Table XIV for any given cDNA clone also may contain one or moreadditional plasmids, each comprising a cDNA clone different from thatgiven clone. Thus, deposits sharing the same ATCC Deposit Number containat least a plasmid for each cDNA clone identified in Table XIV.Typically, each ATCC deposit sample cited in Table XIV comprises amixture of approximately equal amounts (by weight) of about 50 plasmidDNAs, each containing a different cDNA clone; but such a deposit samplemay include plasmids for more or less than 50 cDNA clones, up to about500 cDNA clones.

Two approaches can be used to isolate a particular clone from thedeposited sample of plasmid DNAs cited for that clone in Table XIV.First, a plasmid is directly isolated by screening the clones using apolynucleotide probe corresponding to SEQ ID NO:X.

Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host, as indicated above (such asXL-1Blue (Stratagene)) using techniques known to those of skill in theart, such as those provided by the vector supplier or in relatedpublications or patents cited above. The transformants are plated on1.5% agar plates (containing the appropriate selection agent, e.g.,ampicillin) to a density of about 150 transformants (colonies) perplate. These plates are screened using Nylon membranes according toroutine methods for bacterial colony screening (e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edit., (1989), Cold SpringHarbor Laboratory Press, pages 1.93 to 1.104), or other techniques knownto those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO:X (i.e., within the region of SEQ ID NO:X bounded bythe 5′ NT and the 3′ NT of the clone defined in Table XIV) aresynthesized and used to amplify the desired cDNA using the depositedcDNA plasmid as a template. The polymerase chain reaction is carried outunder routine conditions, for instance, in 25 ul of reaction mixturewith 0.5 ug of the above cDNA template. A convenient reaction mixture is1.5-5 mM MgCl₂, 0.01% (w/v) gelatin, 20 uM each of dATP, dCTP, dGTP,dTTP, 25 pmol of each primer and 0.25 Unit of Taq polymerase. Thirtyfive cycles of PCR (denaturation at 94 degree C. for 1 min; annealing at55 degree C. for 1 min; elongation at 72 degree C. for 1 min) areperformed with a Perkin-Elmer Cetus automated thermal cycler. Theamplified product is analyzed by agarose gel electrophoresis and the DNAband with expected molecular weight is excised and purified. The PCRproduct is verified to be the selected sequence by subcloning andsequencing the DNA product.

Several methods are available for the identification of the 5′ or 3′non-coding portions of a gene which may not be present in the depositedclone. These methods include but are not limited to, filter probing,clone enrichment using specific probes, and protocols similar oridentical to 5′ and 3′ “RACE” protocols which are well known in the art.For instance, a method similar to 5′ RACE is available for generatingthe missing 5′ end of a desired full-length transcript. (Fromont-Racineet al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of thegene of interest is used to PCR amplify the 5′ portion of the desiredfull-length gene. This amplified product may then be sequenced and usedto generate the full length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+RNA can be used. The RNA preparation can then betreated with phosphatase if necessary to eliminate 5′ phosphate groupson degraded or damaged RNA which may interfere with the later RNA ligasestep. The phosphatase should then be inactivated and the RNA treatedwith tobacco acid pyrophosphatase in order to remove the cap structurepresent at the 5′ ends of messenger RNAs. This reaction leaves a 5′phosphate group at the 5′ end of the cap cleaved RNA which can then beligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the desired gene.

Example 2 Isolation of Genomic Clones Corresponding to a Polynucleotide

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCRusing primers selected for the cDNA sequence corresponding to SEQ IDNO:X., according to the method described in Example 1. (See also,Sambrook.)

Example 3 Tissue Distribution of Polypeptide

Tissue distribution of mRNA expression of polynucleotides of the presentinvention is determined using protocols for Northern blot analysis,described by, among others, Sambrook et al. For example, a cDNA probeproduced by the method described in Example 1 is labeled with P³² usingthe rediprime™ DNA labeling system (Amersham Life Science), according tomanufacturer's instructions. After labeling, the probe is purified usingCHROMA SPIN-100™ column (Clontech Laboratories, Inc.), according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) (Clontech) are examined with thelabeled probe using ExpressHyb™ hybridization solution (Clontech)according to manufacturer's protocol number PT1190-1. Followinghybridization and washing, the blots are mounted and exposed to film at−70 degree C. overnight, and the films developed according to standardprocedures.

Example 4 Chromosomal Mapping of the Polynucleotides

An oligonucleotide primer set is designed according to the sequence atthe 5′ end of SEQ ID NO:X. This primer preferably spans about 100nucleotides. This primer set is then used in a polymerase chain reactionunder the following set of conditions: 30 seconds,95 degree C.; 1minute, 56 degree C.; 1 minute, 70 degree C. This cycle is repeated 32times followed by one 5 minute cycle at 70 degree C. Human, mouse, andhamster DNA is used as template in addition to a somatic cell hybridpanel containing individual chromosomes or chromosome fragments (Bios,Inc). The reactions is analyzed on either 8% polyacrylamide gels or 3.5%agarose gels. Chromosome mapping is determined by the presence of anapproximately 100 bp PCR fragment in the particular somatic cell hybrid.

Example 5 Bacterial Expression of a Polypeptide

A polynucleotide encoding a polypeptide of the present invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Amp^(r)), a bacterial origin of replication(ori), an IPTG-regulatable promoter/operator (P/O), a ribosome bindingsite (RBS), a 6-histidine tag (6-His), and restriction enzyme cloningsites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, which expresses the lad repressorand also confers kanamycin resistance (Kan^(r)). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalactopyranoside) is then added to a final concentration of 1 mM. IPTG inducesby inactivating the lad repressor, clearing the P/O leading to increasedgene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4 degree C. The cell debris is removed by centrifugation, and thesupernatant containing the polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni—NTA”) affinity resin column(available from QIAGEN, Inc., supra). Proteins with a 6× His tag bind tothe Ni—NTA resin with high affinity and can be purified in a simpleone-step procedure (for details see: The QIAexpressionist (1995) QIAGEN,Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein can be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins are eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4 degree C. or frozen at −80 degree C.

In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a polynucleotide of the presentinvention, called pHE4a. (ATCC Accession Number 209645, deposited onFeb. 25, 1998.) This vector contains: 1) a neomycinphosphotransferasegene as a selection marker, 2) an E. coli origin of replication, 3) a T5phage promoter sequence, 4) two lac operator sequences, 5) aShine-Delgamo sequence, and 6) the lactose operon repressor gene(lacIq). The origin of replication (oriC) is derived from pUC19 (LTI,Gaithersburg, Md.). The promoter sequence and operator sequences aremade synthetically.

DNA can be inserted into the pHEa by restricting the vector with NdeIand XbaI, BamHI, XhoI, or Asp718, running the restricted product on agel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

The engineered vector could easily be substituted in the above protocolto express protein in a bacterial system.

Example 6 l Purification of a Polypeptide from an Inclusion Body

The following alternative method can be used to purify a polypeptideexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10 degree C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10 degree C. and the cells harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4 degree C. overnight to allowfurther GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stining. The refolded dilutedprotein solution is kept at 4 degree C. without mixing for 12 hoursprior to further purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the polypeptide are then pooled and mixed with 4volumes of water. The diluted sample is then loaded onto a previouslyprepared set of tandem columns of strong anion (Poros HQ-50, PerseptiveBiosystems) and weak anion (Poros CM-20, Perseptive Biosystems) exchangeresins. The columns are equilibrated with 40 mM sodium acetate, pH 6.0.Both columns are washed with 40 mM sodium acetate, pH 6.0, 200 mM NaCl.The CM-20 column is then eluted using a 10 column volume linear gradientranging from 0.2 M NaCl, 50 mM sodium acetate, pH 6.0 to 1.0 M NaCl, 50mM sodium acetate, pH 6.5. Fractions are collected under constant A₂₈₀monitoring of the effluent. Fractions containing the polypeptide(determined, for instance, by 16% SDS-PAGE) are then pooled.

The resultant polypeptide should exhibit greater than 95% purity afterthe above refolding and purification steps. No major contaminant bandsshould be observed from Commassie blue stained 16% SDS-PAGE gel when 5ug of purified protein is loaded. The purified protein can also betested for endotoxin/LPS contamination, and typically the LPS content isless than 0.1 ng/ml according to LAL assays.

Example 7 Cloning and Exression of a Potypt in a Baculorirus ExressionSystem

In this example, the plasmid shuttle vector pA2 is used to insert apolynucleotide into a baculovirus to express a polypeptide. Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI, XbaI and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pAc373, pVL941, and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

Specifically, the cDNA sequence contained in the deposited clone,including the AUG initiation codon and the naturally associated leadersequence identified in Table XIV, is amplified using the PCR protocoldescribed in Example 1. If the naturally occurring signal sequence isused to produce the secreted protein, the pA2 vector does not need asecond signal peptide. Alternatively, the vector can be modified (pA2GP) to include a baculovirus leader sequence, using the standard methodsdescribed in Summers et al., “A Manual of Methods for BaculovirusVectors and Insect Cell Culture Procedures,” Texas AgriculturalExperimental Station Bulletin No. 1555 (1987).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transfectedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Feigner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4 degree C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced protein.

Example 8 Expression of a Polypeptide in Mammalian Cells

The polypeptide of the present invention can be expressed in a mammaliancell. A typical mammalian expression vector contains a promoter element,which mediates the initiation of transcription of mRNA, a protein codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, the polypeptide can be expressed in stable cell linescontaining the polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as dhfr, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulin developing cell lines that carry several hundred or even severalthousand copies of the gene of interest. (See, e.g., Alt, F. W., et al.,J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem.et Biophys. Acta, 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68 (1991).) Another useful selection marker is theenzyme glutamine synthase (GS) (Murphy et al., Biochem J. 227:277-279(1991); Bebbington et al., Bio/Technology 10:169-175 (1992). Using thesemarkers, the mammalian cells are grown in selective medium and the cellswith the highest resistance are selected. These cell lines contain theamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) and NSO cells are often used for the production of proteins.

Derivatives of the plasmid pSV2-dhfr (ATCC Accession No. 37146), theexpression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No.209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985). Multiply cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and asp718, facilitate the cloning ofthe gene of interest. The vectors also contain the 3′ intron, thepolyadenylation and termination signal of the rat preproinisulin gene,and the mouse DHFR gene under control of the SV40 early promoter.

Specifically, the plasmid pC6, for example, is digested with appropriaterestriction enzymes and then dephosphorylated using calf intestinalphosphates by procedures known in the art. The vector is then isolatedfrom a 1% agarose gel.

A polynucleotide of the present invention is amplified according to theprotocol outlined in Example 1. If the naturally occurring signalsequence is used to produce the secreted protein, the vector does notneed a second signal peptide. Alternatively, if the naturally occurringsignal sequence is not used, the vector can be modified to include aheterologous signal sequence. (See, e.g., WO 96/34891.)

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The amplified fragment is then digested with the same restriction enzymeand purified on a 1% agarose gel. The isolated fragment and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC6 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransfection. Five μg of the expression plasmid pC6 a pC4 iscotransfected with 0.5 ug of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).The same procedure is repeated until clones are obtained which grow at aconcentration of 100-200 uM. Expression of the desired gene product isanalyzed, for instance, by SDS-PAGE and Western blot or by reversedphase HPLC analysis.

Example 9 Protein Fusions

The polypeptides of the present invention are preferably fused to otherproteins. These fusion proteins can be used for a variety ofapplications. For example, fusion of the present polypeptides toHis-tag, HA-tag, protein A, IgG domains, and maltose binding proteinfacilitates purification. (See Example 5; see also EP A 394,827;Traunecker, et al., Nature 331:84-86 (1988).) Similarly, fusion toIgG-1, IgG-3, and albumin increases the halflife time in vivo. Nuclearlocalization signals fused to the polypeptides of the present inventioncan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inExample 5.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portioncan be ligated into the BamHI cloning site. Note that the 3′ BamHI siteshould be destroyed. Next, the vector containing the human Fc portion isre-restricted with BamHI, linearizing the vector, and a polynucleotideof the present invention, isolated by the PCR protocol described inExample 1, is ligated into this BamHI site. Note that the polynucleotideis cloned without a stop codon, otherwise a fusion protein will not beproduced.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc region:

       GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAATTCGAGG(SEQ ID NO: 1)GTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGCGACTCTAGAGGAT

Example 10 Production of an Antibody from a Polypeptide

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing a polypeptide of the present invention isadministered to an animal to induce the production of sera containingpolyclonal antibodies. In a preferred method, a preparation of thesecreted protein is prepared and purified to render it substantiallyfree of natural contaminants. Such a preparation is then introduced intoan animal in order to produce polyclonal antisera of greater specificactivity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or protein binding fragments thereof). Suchmonoclonal

antibodies can be prepared using hybridoma technology. (Kohler et al.,Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976);Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., in:Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., pp. 563-681(1981).) In general, such procedures involve immunizing an animal(preferably a mouse) with polypeptide or, more preferably, with asecreted polypeptide-expressing cell. Such cells may be cultured in anysuitable tissue culture medium; however, it is preferable to culturecells in Earle's modified Eagle's medium supplemented with 10% fetalbovine serum (inactivated at about 56 degrees C.), and supplemented withabout 10 g/l of nonessential amino acids, about 1,000 U/ml ofpenicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP2O), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the polypeptide.

Alternatively, additional antibodies capable of binding to thepolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the protein-specificantibody can be blocked by the polypeptide. Such antibodies compriseanti-idiotypic antibodies to the protein-specific antibody and can beused to immunize an animal to induce formation of furtherprotein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, secretedprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

Example 11 Production of Secreted Protein for High-Throughput ScreeningAssays

The following protocol produces a supernatant containing a polypeptideto be tested. This supernatant can then be used in the Screening Assaysdescribed in Examples 13-20.

First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution(1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516 FBiowhittaker) for a working solution of 50 ug/ml. Add 200 ul of thissolution to each well (24 well plates) and incubate at RT for 20minutes. Be sure to distribute the solution over each well (note: a12-channel pipetter may be used with tips on every other channel).Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS(Phosphate Buffered Saline). The PBS should remain in the well untiljust prior to plating the cells and plates may be poly-lysine coated inadvance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in0.5 ml DMEM(Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose andL-glutamine (12-604 F Biowhittaker))/10% heat inactivated FBS(14-503 FBiowhittaker)/1× Penstrep (17-602 E Biowhittaker). Let the cells growovernight.

The next day, mix together in a sterile solution basin: 300 ulLipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem 1 (31985070Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter,aliquot approximately 2 ug of an expression vector containing apolynucleotide insert, produced by the methods described in Examples 8or 9, into an appropriately labeled 96-well round bottom plate. With amulti-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixtureto each well. Pipette up and down gently to mix. Incubate at RT 15-45minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lackingan insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming thefollowing tasks. By tag-teaming, hands on time is cut in half, and thecells do not spend too much time on PBS. First, person A aspirates offthe media from four 24-well plates of cells, and then person B rinseseach well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, andperson B, using a 12-channel pipetter with tips on every other channel,adds the 200 ul of DNA/Lipofectamine/Optimem 1 complex to the odd wellsfirst, then to the even wells, to each row on the 24-well plates.Incubate at 37 degrees C. for 6 hours.

While cells are incubating, prepare appropriate media, either 1 % BSA inDMEM with 1× penstrep, or CHO-5 media (116.6 mg/L of CaCl2 (anhyd);0.00130 mg/L CuSO₄-5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L ofFeSO₄-7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L ofMgSO₄; 6995.50 mg/L of NaCl; 2400.0 of NaHCO₃; 62.50 mg/L ofNaH₂PO₄—H₂O; 71.02 mg/L of Na₂HPO4; 0.4320 mg/L of ZnSO₄-7H₂O; 0.002mg/L of Anachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L ofDL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L ofLinolenic Acid 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid;0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L ofPluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml ofL-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-AsparticAcid; 29.56 mg/ml of L-Cystine-2HCL-H₂O; 31.29 mg/ml of L-Cystine-2HCL;7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/mlof Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml ofL-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL;32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/mlof L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H ₂O; 99.65mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-CaPantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid;15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L ofPyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; and 0.680 mg/L ofVitamin B₁₂; 25 mM of HEPES Buffer, 2.39 mg/L; of Na Hypoxanthine; 0.105mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L ofSodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 2 uM of Ethanolamnine;0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrincomplexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrincomplexed with Oleic Acid; and 10 mg/L of Methyl-B-Cyclodextrincomplexed with Retinal) with 2 mm glutamine and 1× penstrep. (BSA(81-068-3 Bayer) 100 gm dissolved in 1L DMEM for a 10% BSA stocksolution). Filter the media and collect 50 ul for endotoxin assay in 15ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, atthe end of the incubation period. Person A aspirates off thetransfection media, while person B adds 1.5 ml appropriate media to eachwell. Incubate at 37 degrees C. for 45 or 72 hours depending on themedia used: 1% BSA for 45 hours or CHO-5 for 72 hours.

On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one1 ml deep well plate and the remaining supernatant into a 2 ml deepwell. The supernatants from each well can then be used in the assaysdescribed in Examples 13-20.

It is specifically understood that when activity is obtained in any ofthe assays described below using a supernatant, the activity originatesfrom either the polypeptide directly (e.g., as a secreted protein) or bythe polypeptide inducing expression of other proteins, which are thensecreted into the supernatant. Thus, the invention further provides amethod of identifying the protein in the supernatant characterized by anactivity in a particular assay.

Example 12 Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation andproliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factorscalled Signal Transducers and Activators of Transcription, or “STATs.”There are six members of the STATs family. Stat1 and Stat3 are presentin many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleusupon tyrosine phosphorylation by a set of kinases known as the JanusKinase (“Jaks”) family. Jaks represent a distinct family of solubletyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinasesdisplay significant sequence similarity and are generally catalyticallyinactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in theTable below. (Adapted from review by Schidler and Damell, Ann. Rev.Biochem. 64:621-51 (1995).) A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proximal region encodingTrp-Ser-Xxx-Trp-Ser (SEQ ID NO:2)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which inturn activate STATs, which then translocate and bind to GAS elements.This entire process is encompassed in the Jaks-STATs signal transductionpathway.

Therefore, activation of the Jaks-STATs pathway, reflected by thebinding of the GAS or the ISRE element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells. For example,growth factors and cytokines are known to activate the Jaks-STATspathway. (See Table below.) Thus, by using GAS elements linked toreporter molecules, activators of the Jaks-STATs pathway can beidentified.

JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS(elements) or ISRE IFN familyIFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 > IFP)Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotrophic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP) Il-11(Pleiotrophic) ? + ? ? 1, 3 OnM(Pleiotrophic)? + + ? 1, 3 LIF(Pleiotrophic) ? + + ? 1, 3 CNTF(Pleiotrophic) −/+ + + ?1, 3 G-CSF(Pleiotrophic) ? + ? ? 1, 3 IL-12(Pleiotrophic) + − + + 1, 3g-C family IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid)− + − + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GASIL-9 (lymphocytes) − + − + 5 GAS IL-13 (lymphocyte) − + ? ? 6 GAS IL-15? + ? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >>Ly6) IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5GAS(B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1,3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element, which is usedin the Biological Assays described in Examples 13-14, a PCR basedstrategy is employed to generate a GAS-SV40 promoter sequence. The 5′primer contains four tandem copies of the GAS binding site found in theIRF1 promoter and previously demonstrated to bind STATs upon inductionwith a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).),although other GAS or ISRE elements can be used instead. The 5′ primeralso contains 18 bp of sequence complementary to the SV40 early promotersequence and is flanked with an XhoI site. The sequence of the 5′ primeris:

5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCC(SEQ ID NO: 3) ATCTCAATTAG:3′

The downstream primer is complementary to the SV40 promoter and isflanked with a Hind III site:

5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 4)

PCR amplification is performed using the SV40 promoter template presentin the B-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI/Hind III and subcloned into BLSK2-.(Stratagene.) Sequencing with forward and reverse primers confirms thatthe insert contains the following sequence:

     5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAAATGATTTCCCCGAAATGATTTCCCCGAAATATCTGC(SEQ ID NO: 5)CATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2reporter construct is next engineered. Here, the reporter molecule is asecreted alkaline phosphatase, or “SEAP.” Clearly, however, any reportermolecule can be instead of SEAP, in this or in any of the otherExamples. Well known reporter molecules that can be used instead of SEAPinclude chloramphenicol acetyltransferase (CAT), luciferase, alkalinephosphatase, B-galactosidase, green fluorescent protein (GFP), or anyprotein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element issubcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing theGAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAPvector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 13-14.

Other constructs can be made using the above description and replacingGAS with a different promoter sequence. For example, construction ofreporter molecules containing NFK-B and EGR promoter sequences aredescribed in Examples 15 and 16. However, many other promoters can besubstituted using the protocols described in these Examples. Forinstance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted,alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, II-2/NFAT, orNF-KB/GAS). Similarly, other cell lines can be used to test reporterconstruct activity, such as HELA (epithelial), HUVEC (endothelial), Reh(B-cell), Saos-2 (osteoblast), HUTVAC (aortic), or Cardiomyocyte.

Example 13 High-Throughput Screening Assay for T-Cell Activity

The following protocol is used to assess T-cell activity by identifyingfactors, and determining whether supernate containing a polypeptide ofthe invention proliferates and/or differentiates T-cells. T-cellactivity is assessed using the GAS/SEAP/Neo construct produced inExample 12. Thus, factors that increase SEAP activity indicate theability to activate the Jaks-STATS signal transduction pathway. TheT-cell used in this assay is Jurkat T-cells (ATCC Accession No.TIB-152), although Molt-3 cells (ATCC Accession No. CRL-1552) and Molt-4cells (ATCC Accession No. CRL-1582) cells can also be used.

Jurkat T-cells are lymphoblastic CD4+ Th1 helper cells. In order togenerate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

Specifically, the following protocol will yield sufficient cells for 75wells containing 200 ul of cells. Thus, it is either scaled up, orperformed in multiple to generate sufficient cells for multiple 96 wellplates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep.Combine 2.5 mis of OPTI-MEM (Life Technologies) with 10 ug of plasmidDNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C andincubate at room temperature for 15-45 mins.

During the incubation period, count cell concentration, spin down therequired number of cells (10⁷ per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37 degrees C. for 6hrs. After the incubation, add 10 ml of RPMI+15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10%serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated withsupernatants containing polypeptides of the invention and/or inducedpolypeptides of the invention as produced by the protocol described inExample 11.

On the day of treatment with the supernatant, the cells should be washedand resuspended in fresh RPMI+10% serum to a density of 500,000 cellsper ml. The exact number of cells required will depend on the number ofsupernatants being screened. For one 96 well plate, approximately 10million cells (for 10 plates, 100 million cells) are required.

Transfer the cells to a triangular reservoir boat, in order to dispensethe cells into a 96 well dish, using a 12 channel pipette. Using a 12channel pipette, transfer 200 ul of cells into each well (thereforeadding 100,000 cells per well).

After all the plates have been seeded, 50 ul of the supernatants aretransferred directly from the 96 well plate containing the supernatantsinto each well using a 12 channel pipette. In addition, a dose ofexogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10,and H11 to serve as additional positive controls for the assay.

The 96 well dishes containing Jurkat cells treated with supernatants areplaced in an incubator for 48 hrs (note: this time is variable between48-72 hrs). 35 ul samples from each well are then transferred to anopaque 96 well plate using a 12 channel pipette. The opaque platesshould be covered (using sellophene covers) and stored at −20 degrees C.until SEAP assays are performed according to Example 17. The platescontaining the remaining treated cells are placed at 4 degrees C. andserve as a source of material for repeating the assay on a specific wellif desired.

As a positive control, 100 Unit/ml interferon gamma can be used which isknown to activate Jurkat T cells. Over 30 fold induction is typicallyobserved in the positive control wells.

The above protocol may be used in the generation of both transient, aswell as, stable transfected cells, which would be apparent to those ofskill in the art.

Example 14 High-Throughput Screening Assay Identifying Myeloid Activity

The following protocol is used to assess myeloid activity by determiningwhether polypeptides of the invention proliferates and/or differentiatesmyeloid cells. Myeloid cell activity is assessed using the GAS/SEAP/Neoconstruct produced in Example 12. Thus, factors that increase SEAPactivity indicate the ability to activate the Jaks-STATS signaltransduction pathway. The myeloid cell used in this assay is U937, apre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

To transiently transfect U937 cells with the GAS/SEAP/Neo constructproduced in Example 12, a DEAE-Dextran method (Kharbanda et. al., 1994,Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10e⁷U 937 cells and wash with PBS. The U937 cells are usually grown in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS)supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂O, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37 degrees C. for 45 min.

Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37 degrees C. for 36hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400ug/ml G418. The G418-free medium used for routine growth but every oneto two months, the cells should be re-grown in 400 ug/ml G418 for coupleof passages.

These cells are tested by harvesting 1×10⁸ cells (this is enough for ten96-well plates assay) and wash with PBS. Suspend the cells in 200 mlabove described growth medium, with a final density of 5×10⁵ cells/ml.Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).

Add 50 ul of the supernatant prepared by the protocol described inExample 11. Incubate at 37 degrees C. for 48 to 72 hr. As a positivecontrol, 100 Unit/ml interferon gamma can be used which is known toactivate U937 cells. Over 30 fold induction is typically observed in thepositive control wells. SEAP assay the supernatant according to theprotocol described in Example 17.

Example 15 High-Throughput Screening Assay Identifying Neuronal Activity

When cells undergo differentiation and proliferation, a group of genesare activated through many different signal transduction pathways. Oneof these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, activation of cells can be assessed.

Particularly, the following protocol is used to assess neuronal activityin PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are knownto proliferate and/or differentiate by activation with a number ofmitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growthfactor), and EGF (epidermal growth factor). The EGR1 gene expression isactivated during this treatment. Thus, by stably transfecting PC12 cellswith a construct containing an EGR promoter linked to SEAP reporter,activation of PC12 cells can be assessed.

The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers:

5′ (SEQ ID NO: 6) GCGCTCGAGGGATGACAGCGATAGAACCCCGG-3′ 5′ (SEQ ID NO: 7)GCGAAGCTTCGCGACTCCCCGGATCCGCCTC-3′

Using the GAS:SEAP/Neo vector produced in Example 12, EGR1 amplifiedproduct can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mis of a coatingsolution (1:30 dilution of collagen type 1 (Upstate Biotech Inc.Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5%heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/mlpenicillin and 100 ug/ml streptomycin on a precoated 10 cm tissueculture dish. One to four split is done every three to four days. Cellsare removed from the plates by scraping and resuspended with pipettingup and down for more than 15 times.

Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamineprotocol described in Example 11. EGR-SEAP/PC12 stable cells areobtained by growing the cells in 300 ug/ml G418. The G418-free medium isused for routine growth but every one to two months, the cells should bere-grown in 300 ug/ml G418 for couple of passages.

To assay for neuronal activity, a 10 cm plate with cells around 70 to80% confluent is screened by removing the old medium. Wash the cellsonce with PBS (Phosphate buffered saline). Then starve the cells in lowserum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS withantibiotics) overnight.

The next morning, remove the medium and wash the cells with PBS. Scrapeoff the cells from the plate, suspend the cells well in 2 ml low serummedium. Count the cell number and add more low serum medium to reachfinal cell density as 5×10⁵ cells/ml.

Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×10⁵ cells/well). Add 50 ul supernatant produced byExample 11, 37° C. for 48 to 72 hr. As a positive control, a growthfactor known to activate PC12 cells through EGR can be used, such as 50ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAPis typically seen in the positive control wells. SEAP assay thesupernatant according to Example 17.

Example 16 High-Throughput Screening Assay for T-Cell Activity

NF-KB (Nuclear Factor KB) is a transcription factor activated by a widevariety of agents including the inflammatory cytokines IL-1 and TNF,CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposure toLPS or thrombin, and by expression of certain viral gene products. As atranscription factor, NF-KB regulates the expression of genes involvedin immune cell activation, control of apoptosis (NF-KB appears to shieldcells from apoptosis), B and T-cell development, anti-viral andantimicrobial responses, and multiple stress responses.

In non-stimulated conditions, NF-KB is retained in the cytoplasm withI-KB (Inhibitor KB). However, upon stimulation, I-KB is phosphorylatedand degraded, causing NF-KB to shuttle to the nucleus, therebyactivating transcription of target genes. Target genes activated byNF-KB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1 MHC.

Due to its central role and ability to respond to a range of stimuli,reporter constructs utilizing the NF-KB promoter element are used toscreen the supernatants produced in Example 11. Activators or inhibitorsof NF-KB would be useful in treating diseases. For example, inhibitorsof NF-KB could be used to treat those diseases related to the acute orchronic activation of NF-KB, such as rheumatoid arthritis.

To construct a vector containing the NF-KB promoter element, a PCR basedstrategy is employed. The upstream primer contains four tandem copies ofthe NF-KB binding site (GGGGACTTTCCC) (SEQ ID NO:8), 18 bp of sequencecomplementary to the 5′ end of the SV40 early promoter sequence, and isflanked with an XhoI site:

5′:GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCCTGCCATCTCAATTAG:3′(SEQ ID NO: 9)

The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site:

5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ ID NO: 4)

PCR amplification is performed using the SV40 promoter template presentin the pB-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI and Hind III and subcloned into BLSK2-.(Stratagene) Sequencing with the T7 and T3 primers confirms the insertcontains the following sequence:

5′:CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGACTTTCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAAC(SEQ ID NO: 10)CATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTT:3′

Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-KB/SV40 fragment usingXhoI and Hindlll. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

In order to generate stable mammalian cell lines, the NF-KB/SV40/SEAPcassette is removed from the above NF-KB/SEAP vector using restrictionenzymes SalI and NotI, and inserted into a vector containing neomycinresistance. Particularly, the NF-KB/SV40/SEAP cassette was inserted intopGFP-1 (Clontech), replacing the GFP gene, after restricting pGFP-1 withSalI and NotI.

Once NF-KB/SV40/SEAP/Neo vector is created, stable Jurkat T-cells arecreated and maintained according to the protocol described in Example13. Similarly, the method for assaying supernatants with these stableJurkat T-cells is also described in Example 13. As a positive control,exogenous TNF alpha (0.1,1, 10 ng) is added to wells H9, H10, and H11,with a 5-10 fold activation typically observed.

Example 17 Assay for SEAP Activity

As a reporter molecule for the assays described in Examples 13-16, SEAPactivity is assayed using the Tropix Phospho-light Kit (Cat. BP-400)according to the following general procedure. The Tropix Phospho-lightKit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of2.5× dilution buffer into Optiplates containing 35 ul of a supernatant.Seal the plates with a plastic sealer and incubate at 65 degree C. for30 min. Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenserand prime with the Assay Buffer. Add 50 ml assay Buffer and incubate atroom temperature 5 min. Empty the dispenser and prime with the ReactionBuffer (see the table below). Add 50 ul Reaction Buffer and incubate atroom temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on luminometer, one should treat 5 plates at each timeand start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, andprint the results. An increase in chemiluminescence indicates reporteractivity.

Reaction Buffer Formulation: # of plates Rxn buffer diluent (ml) CSPD(ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 85 4.25 16 904.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22 120 6 23125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.5 29 1557.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 185 9.25 36190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 215 10.75 42220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 245 12.25 48250 12.5 49 255 12.75 50 260 13

Example 18 High-Throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levelsof small molecules, such as calcium, potassium, sodium, and pH, as wellas alter membrane potential. These alterations can be measured in anassay to identify supernatants which bind to receptors of a particularcell. Although the following protocol describes an assay for calcium,this protocol can easily be modified to detect changes in potassium,sodium, pH, membrane potential, or any other small molecule which isdetectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) tomeasure changes in fluorescent molecules (Molecular Probes) that bindsmall molecules. Clearly, any fluorescent molecule detecting a smallmolecule can be used instead of the calcium fluorescent molecule, fluo-4(Molecular Probes, Inc.; catalog no. F-14202), used here.

For adherent cells, seed the cells at 10,000-20,000 cells/well in aCo-star black 96-well plate with clear bottom. The plate is incubated ina CO₂ incubator for 20 hours. The adherent cells are washed two times inBiotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

A stock solution of 1 mg/ml fluo-4 is made in 10% pluronic acid DMSO. Toload the cells with fluo-4, 50 ul of 12 ug/ml fluo-4 is added to eachwell. The plate is incubated at 37 degrees C. in a CO₂ incubator for 60min. The plate is washed four times in the Biotek washer with HBSSleaving 100 ul of buffer.

For non-adherent cells, the cells are spun down from culture media.Cells are re-suspended to 2-5×10⁶ cell/ml with HBSS in a 50-ml conicaltube. 4 ul of 1 mg/ml fluo-4 solution in 10% pluronic acid DMSO is addedto each ml of cell suspension. The tube is then placed in a 37 degreesC. water bath for 30-60 min. The cells are washed twice with HBSS,resuspended to 1×10⁶ cells/ml, and dispensed into a microplate, 100ul/well. The plate is centrifuged at 1000 rpm for 5 min. The plate isthen washed once in Denley CellWash with 200 ul, followed by anaspiration step to 100 ul final volume.

For a non-cell based assay, each well contains a fluorescent molecule,such as fluo-4. The supernatant is added to the well, and a change influorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is setfor the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488; (5) Emission is 530 nm; and (6) Sample addition is 50 ul. Increasedemission at 530 nm indicates an extracellular signaling event which hasresulted in an increase in the intracellular Ca⁺⁺ concentration.

Example 19 High-Throughput Screening Assay Identifying Tyrosine KinaseActivity

The Protein Tyrosine Kinases (PTK) represent a diverse group oftrarsmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, lck, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, the identification of novel human secretedproteins capable of activating tyrosine kinase signal transductionpathways are of interest. Therefore, the following protocol is designedto identify those novel human secreted proteins capable of activatingthe tyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type 1 collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4 degree C. Cell growth on these plates is assayed by seeding 5,000cells/well in growth medium and indirect quantitation of cell numberthrough use of alamarBlue as described by the manufacturer AlamarBiosciences, Inc. (Sacramento, Calif.) after 48 hr. Falcon plate covers#3071 from Becton Dickinson (Bedford, Mass.) are used to cover theLoprodyne Silent Screen Plates. Falcon Microtest III cell culture platescan also be used in some proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes ofLoprodyne plates (20,000/200 ml/well) and cultured overnight in completemedium. Cells are quiesced by incubation in serum-free basal medium for24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of thesupernatant produced in Example 11, the medium was removed and 100 ml ofextraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100,0.1% SDS, 2 mM Na3VO4, 2 mM Na4P2O7 and a cocktail of proteaseinhibitors (#1836170) obtained from Boeheringer Mannheim (Indianapolis,Ind.) is added to each well and the plate is shaken on a rotating shakerfor 5 minutes at 4 degrees C. The plate is then placed in a vacuumtransfer manifold and the extract filtered through the 0.45 mm membranebottoms of each well using house vacuum. Extracts are collected in a96-well catch/assay plate in the bottom of the vacuum manifold andimmediately placed on ice. To obtain extracts clarified bycentrifugation, the content of each well, after detergent solubilizationfor 5 minutes, is removed and centrifuged for 15 minutes at 4 degrees C.at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity.Although many methods of detecting tyrosine kinase activity are known,one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated bydetermining its ability to phosphorylate a tyrosine residue on aspecific substrate (a biotinylated peptide). Biotinylated peptides thatcan be used for this purpose include PSK1 (corresponding to amino acids6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding toamino acids 1-17 of gastrin). Both peptides are substrates for a rangeof tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate(1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30 degrees C. for 2 min. Initial thereaction by adding 10 ul of the control enzyme or the filteredsupernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot ofreaction mixture to a microtiter plate (MTP) module and incubating at 37degrees C. for 20 min. This allows the streptavadin coated 96 well plateto associate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul of anti-phospotyrosineantibody conjugated to horse radish peroxidase(anti-P-Tyr-POD(0.5 u/ml))to each well and incubate at 37 degrees C. for one hour. Wash the wellas above.

Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim)and incubate at room temperature for at least 5 mins (up to 30 min).Measure the absorbance of the sample at 405 nm by using ELISA reader.The level of bound peroxidase activity is quantitated using an ELISAreader and reflects the level of tyrosine kinase activity.

Example 20 High-Throughput Screening Assay Identifying PhosphorylationActivity

As a potential alternative and/or compliment to the assay of proteintyrosine kinase activity described in Example 19, an assay which detectsactivation (phosphorylation) of major intracellular signal transductionintermediates can also be used. For example, as described below oneparticular assay can detect tyrosine phosphorylation of the Erk-1 andErk-2 kinases. However, phosphorylation of other molecules, such as Raf,JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specifickinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,phosphotyrosine, or phosphothreonine molecule, can be detected bysubstituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-wellELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp,(RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBSfor 1 hr at RT. The protein G plates are then treated with 2 commercialmonoclonal antibodies (100 ng/well) against Erk-1 and Erk-2 (1 hr at RT)(Santa Cruz Biotechnology). (To detect other molecules, this step caneasily be modified by substituting a monoclonal antibody detecting anyof the above described molecules.) After 3-5 rinses with PBS, the platesare stored at 4 degrees C. until use.

A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplateand

cultured overnight in growth medium. The cells are then starved for 48hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50 ulof the supernatants obtained in Example 11 for 5-20 minutes. The cellsare then solubilized and extracts filtered directly into the assayplate.

After incubation with the extract for 1 hr at RT, the wells are againrinsed. As a positive control, a commercial preparation of MAP kinase(10 ng/well) is used in place of A431 extract. Plates are then treatedwith a commercial polyclonal (rabbit) antibody (1 ug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFLA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation.

Example 21 Method of Determining Alterations in a Gene Corresponding toa Polynucleotide

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:X. Suggested PCRconditions consist of 35 cycles at 95 degrees C. for 30 seconds; 60-120seconds at 52-58 degrees C.; and 60-120 second at 70 degrees C., usingbuffer solutions described in Sidransky et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons isalso determined and genomic PCR products analyzed to confirm theresults. PCR products harboring suspected mutations is then cloned andsequenced to validate the results of the direct sequencing.

PCR products is cloned into T-tailed vectors as described in Holton etal., Nucleic Acids Research, 19:1156 (1991) and sequenced with T7polymerase (United States Biochemical). Affected individuals areidentified by mutations not present in unaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in a gene corresponding to a polynucleotide. Genomic clonesisolated according to Example 2 are nick-translated withdigoxigenindeoxy-uridine 5′-triphosphate (Boehringer Manheim), and FISHperformed as described in Johnson et al., Methods Cell Biol. 35:73-99(1991). Hybridization with the labeled probe is carried out using a vastexcess of human cot-1 DNA for specific hybridization to thecorresponding genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C— and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region hybridized by the probe are identifiedas insertions, deletions, and translocations. These alterations are usedas a diagnostic marker for an associated disease.

Example 22 Method of Detecting Abnormal Levels of a Polypeptide in aBiological Sample

A polypeptide of the present invention can be detected in a biologicalsample, and if an increased or decreased level of the polypeptide isdetected, this polypeptide is a marker for a particular phenotype.Methods of detection are numerous, and thus, it is understood that oneskilled in the art can modify the following assay to fit theirparticular needs.

For example, antibody-sandwich ELISAs are used to detect polypeptides ina sample, preferably a biological sample. Wells of a microtiter plateare coated with specific antibodies, at a final concentration of 0.2 to10 ug/ml. The antibodies are either monoclonal or polyclonal and areproduced by the method described in Example 10. The wells are blocked sothat non-specific binding of the polypeptide to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining the polypeptide. Preferably, serial dilutions of the sampleshould be used to validate results. The plates are then washed threetimes with deionized or distilled water to remove unbounded polypeptide.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the polypeptide in the sample using the standard curve.

Example 23 Formulation

The invention also provides methods of treatment and/or prevention ofdiseases or disorders (such as, for example, any one or more of thediseases or disorders disclosed herein) by administration to a subjectof an effective amount of a Therapeutic. By therapeutic is meant apolynucleotides or polypeptides of the invention (including fragmentsand variants), agonists or antagonists thereof, and/or antibodiesthereto, in combination with a pharmaceutically acceptable carrier type(e.g., a sterile carrier).

The Therapeutic will be formulated and dosed in a fashion consistentwith good medical practice, taking into account the clinical conditionof the individual patient (especially the side effects of treatment withthe Therapeutic alone), the site of delivery, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” for purposes herein isthus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofthe Therapeutic administered parenterally per dose will be in the rangeof about 1 ug/kg/day to 10 mg/kg/day of patient body weight, although,as noted above, this will be subject to therapeutic discretion. Morepreferably, this dose is at least 0.01 mg/kg/day, and most preferablyfor humans between about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, the Therapeutic is typically administered at a dose rateof about 1 ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injectionsper day or by continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. The lengthof treatment needed to observe changes and the interval followingtreatment for responses to occur appears to vary depending on thedesired effect.

Therapeutics can be are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any. The term “parenteral” as usedherein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics are administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, gels, drops or transdermal patch), bucally, or as anoral or nasal spray. “Pharmaceutically acceptable carrier” refers to anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrastemal, subcutaneous andintraarticular injection and infusion.

Therapeutics of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releaseTherapeutics include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman et al., Biopolymers 22:547-556 (1983)),poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed. Mater.Res. 15:167-277 (1981), and Langer, Chem. Tech. 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Sustained-release Therapeutics also include liposomally entrappedTherapeutics of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing theTherapeutic are prepared by methods known per se: DE 3,218,121; Epsteinet al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al.,Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676;EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S.Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, theliposomes are of the small (about 200-800 Angstroms) unilamellar type inwhich the lipid content is greater than about 30 mol. percentcholesterol, the selected proportion being adjusted for the optimalTherapeutic.

In yet an additional embodiment, the Therapeutics of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. RefBiomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, the Therapeutic isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to the Therapeutic.

Generally, the formulations are prepared by contacting the Therapeuticuniformly and intimately with liquid carriers or finely divided solidcarriers or both. Then, if necessary, the product is shaped into thedesired formulation. Preferably the carrier is a parenteral carrier,more preferably a solution that is isotonic with the blood of therecipient. Examples of such carrier vehicles include water, saline,Ringer's solution, and dextrose solution. Non-aqueous vehicles such asfixed oils and ethyl oleate are also useful herein, as well asliposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmarmitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

The Therapeutic is typically formulated in such vehicles at aconcentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, ata pH of about 3 to 8. It will be understood that the use of certain ofthe foregoing excipients, carriers, or stabilizers will result in theformation of polypeptide salts.

Any pharmaceutical used for therapeutic administration can be sterile.Sterility is readily accomplished by filtration through sterilefiltration membranes (e.g., 0.2 micron membranes). Therapeuticsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

Therapeutics ordinarily will be stored in unit or multi-dose containers,for example, sealed ampoules or vials, as an aqueous solution or as alyophilized formulation for reconstitution. As an example of alyophilized formulation, 10-ml vials are filled with 5 ml ofsterile-filtered 1% (w/v) aqueous Therapeutic solution, and theresulting mixture is lyophilized. The infusion solution is prepared byreconstituting the lyophilized Therapeutic using bacteriostaticWater-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of theTherapeutics of the invention. Associated with such container(s) can bea notice in the form prescribed by a governmental agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration. In addition, the Therapeutics may be employedin conjunction with other therapeutic compounds.

The Therapeutics of the invention may be administered alone or incombination with adjuvants. Adjuvants that may be administered with theTherapeutics of the invention include, but are not limited to, alum,alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21(Genentech, Inc.), BCG, and MPL. In a specific embodiment, Therapeuticsof the invention are administered in combination with alum. In anotherspecific embodiment, Therapeutics of the invention are administered incombination with QS-21. Further adjuvants that may be administered withthe Therapeutics of the invention include, but are not limited to,Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18,CRL1005, Aluminum salts, MF-59, and Virosomal adjuvant technology.Vaccines that may be administered with the Therapeutics of the inventioninclude, but are not limited to, vaccines directed toward protectionagainst MMR (measles, mumps, rubella), polio, varicella,tetanus/diptheria, hepatitis A, hepatitis B, haemophilus influenzae B,whooping cough, pneumonia, influenza, Lyme's Disease, rotavirus,cholera, yellow fever, Japanese encephalitis, poliomyelitis, rabies,typhoid fever, and pertussis. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

The Therapeutics of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the Therapeutics of the invention,include but not limited to, other members of the TNF family,chemotherapeutic agents, antibiotics, steroidal and non-steroidalanti-inflammatories, conventional immunotherapeutic agents, cytokinesand/or growth factors. Combinations may be administered eitherconcomitantly, e.g., as an admixture, separately but simultaneously orconcurrently; or sequentially. This includes presentations in which thecombined agents are administered together as a therapeutic mixture, andalso procedures in which the combined agents are administered separatelybut simultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In one embodiment, the Therapeutics of the invention are administered incombination with members of the TNF family. TNF, TNF-related or TNF-likemolecules that may be administered with the Therapeutics of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880), TR6(International Publication No. WO 98/30694), OPG, and neutrokine-alpha(International Publication No. WO 98/18921, OX40, and nerve growthfactor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, TR2(International Publication No. WO 96/34095), DR3 (InternationalPublication No. WO 97/33904), DR4 (International Publication No. WO98/32856), TR5 (International Publication No. WO 98/30693), TR6(International Publication No. WO 98/30694), TR7 (InternationalPublication No. WO 98/41629), TRANK, TR9 (International Publication No.WO 98/56892),TR10 (International Publication No. WO 98/54202), 312C2(International Publication No. WO 98/06842), and TR12, and soluble formsCD154, CD70, and CD153.

In certain embodiments, Therapeutics of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the Therapeuticsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (larnivudine/3TC), and COMBIVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the Therapeutics of theinvention, include, but are not limited to, CRDCIVAN™ (indinavir),NORVIR™ (ritonavir), INWIRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith Therapeutics of the invention to treat AIDS and/or to prevent ortreat HIV infection.

In other embodiments, Therapeutics of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe Therapeutics of the invention, include, but are not limited to,TRIDETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, Therapeutics of theinvention are used in any combination withTRBMTHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, and/orATOVAQUONE™ to prophylactically treat or prevent an opportunisticPneumocystis carinii pneumonia infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, and/or ETHAMBUTOL™ toprophylactically treat or prevent an opportunistic Mycobacterium aviumcomplex infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with RIFABUTIN™, CLARITHROMYCIN™,and/or AZITHROMYCIN™ to prophylactically treat or prevent anopportunistic Mycobacterium tuberculosis infection. In another specificembodiment, Therapeutics of the invention are used in any combinationwith GANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylacticallytreat or prevent an opportunistic cytomegalovirus infection. In anotherspecific embodiment, Therapeutics of the invention are used in anycombination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™ toprophylactically treat or prevent an opportunistic fungal infection. Inanother specific embodiment, Therapeutics of the invention are used inany combination with ACYCLOVIR™ and/or FAMCICOLVIR™ to prophylacticallytreat or prevent an opportunistic herpes simplex virus type I and/ortype II infection. In another specific embodiment, Therapeutics of theinvention are used in any combination with PYRIMETHAMINE™ and/orLEUCOVORIN™ to prophylactically treat or prevent an opportunisticToxoplasma gondli infection. In another specific embodiment,Therapeutics of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat or prevent anopportunistic bacterial infection.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the Therapeutics of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the Therapeutics of the invention include,but are not limited to, amoxicillin, beta-lactamases, aminoglycosides,beta-lactam (glycopeptide), beta-lactamases, Clindamycin,chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin,erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins,quinolones, rifampin, streptomycin, sulfonamide, tetracyclines,trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the Therapeutics of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs, cyclophosphamide methylprednisone, prednisone, azathioprine,FK-506, 15-deoxyspergualin, and other immunosuppressive agents that actby suppressing the function of responding T cells.

In specific embodiments, Therapeutics of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the Therapeutics of the invention include,but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMNE™/NEORAL™/SANGDYA™(cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In an additional embodiment, Therapeutics of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the Therapeutics of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, Therapeutics of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In an additional embodiment, the Therapeutics of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the Therapeuticsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compositions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the Therapeutics of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, Therapeutics of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, Therapeutics of the invention are administered incombination with Rituximab. In a further embodiment, Therapeutics of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the Therapeutics of the invention include, but are notlimited to, IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL12, IL13, IL15,anti-CD40, CD40L, IFN-gamma and TNF-alpha. In another embodiment,Therapeutics of the invention may be administered with any interleukin,including, but not limited to, IL-1 alpha, IL-1beta, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18, IL-19, IL-20, and IL-21.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the Therapeutics of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B (VEGF-3); Vascular Endothelial Growth Factor B-186(VEGF-B186), as disclosed in International Publication Number WO96/26736; Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/02543; Vascular EndothelialGrowth Factor-D (VEGF-D), as disclosed in International PublicationNumber WO 98/07832; and Vascular Endothelial Growth Factor-E (VEGF-E),as disclosed in German Patent Number DE19639601. The above mentionedreferences are incorporated herein by reference herein.

In an additional embodiment, the Therapeutics of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with theTherapeutics of the invention include, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the Therapeutics of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the Therapeutics of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the Therapeutics of the invention areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Example 24 Method of Treating Decreased Levels of the Polypeptide

The present invention relates to a method for treating an individual inneed of an increased level of a polypeptide of the invention in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of an agonist of the invention(including polypeptides of the invention). Moreover, it will beappreciated that conditions caused by a decrease in the standard ornormal expression level of a secreted protein in an individual can betreated by administering the polypeptide of the present invention,preferably in the secreted form. Thus, the invention also provides amethod of treatment of an individual in need of an increased level ofthe polypeptide comprising administering to such an individual aTherapeutic comprising an amount of the polypeptide to increase theactivity level of the polypeptide in such an individual.

For example, a patient with decreased levels of a polypeptide receives adaily dose 0.1-100 ug/kg of the polypeptide for six consecutive days.Preferably, the polypeptide is in the secreted form. The exact detailsof the dosing scheme, based on administration and formulation, areprovided in Example 23.

Example 25 Method of Treating Increased Levels of the Polypeptide

The present invention also relates to a method of treating an individualin need of a decreased level of a polypeptide of the invention in thebody comprising administering to such an individual a compositioncomprising a therapeutically effective amount of an antagonist of theinvention (including polypeptides and antibodies of the invention).

In one example, antisense technology is used to inhibit production of apolypeptide of the present invention. This technology is one example ofa method of decreasing levels of a polypeptide, preferably a secretedform, due to a variety of etiologies, such as cancer. For example, apatient diagnosed with abnormally increased levels of a polypeptide isadministered intravenously antisense polynucleotides at 0.5, 1.0, 1.5,2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a7-day rest period if the treatment was well tolerated. The formulationof the antisense polynucleotide is provided in Example 23.

Example 26 Method of Treatment Using Gene Therapy-Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing a polypeptide, onto a patient. Generally, fibroblasts areobtained from a subject by skin biopsy. The resulting tissue is placedin tissue-culture medium and separated into small pieces. Small chunksof the tissue are placed on a wet surface of a tissue culture flask,approximately ten pieces are placed in each flask. The flask is turnedupside down, closed tight and left at room temperature over night. After24 hours at room temperature, the flask is inverted and the chunks oftissue remain fixed to the bottom of the flask and fresh media (e.g.,Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added.The flasks are then incubated at 37 degree C. for approximately oneweek.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention can beamplified using PCR primers which correspond to the 5′ and 3′ endsequences respectively as set forth in Example 1 using primers andhaving appropriate restriction sites and initiation/stop codons, ifnecessary. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector has the gene ofinterest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellstransduced with the vector. The packaging cells now produce infectiousviral particles containing the gene (the packaging cells are nowreferred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether protein isproduced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 27 Gene Therapy Using Endogenous Genes Corresponding ToPolynucleotides of the Invention

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous polynucleotide sequence ofthe invention with a promoter via homologous recombination as described,for example, in U.S. Pat. No: 5,641,670, issued Jun. 24, 1997;International Publication No: WO 96/29411, published Sep. 26, 1996;International Publication No: WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad Sci. USA, 86:8932-8935 (1989); andZijlstra et al., Nature, 342:435-438 (1989). This method involves theactivation of a gene which is present in the target cells, but which isnot expressed in the cells, or is expressed at a lower level thandesired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous polynucleotide sequence, flanking the promoter. Thetargeting sequence will be sufficiently near the 5′ end of thepolynucleotide sequence so the promoter will be operably linked to theendogenous sequence upon homologous recombination. The promoter and thetargeting sequences can be amplified using PCR. Preferably, theamplified promoter contains distinct restriction enzyme sites on the 5′and 3′ ends. Preferably, the 3′ end of the first targeting sequencecontains the same restriction enzyme site as the 5′ end of the amplifiedpromoter and the 5′ end of the second targeting sequence contains thesame restriction site as the 3′ end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenouspolynucleotide sequence. This results in the expression ofpolynucleotide corresponding to the polynucleotide in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the locus corresponding to thepolynucleotide of the invention, plasmid pUC18 (MB1 Fennentas, Amherst,N.Y.) is digested with HindIII. The CMV promoter is amplified by PCRwith an XbaI site on the 5′ end and a BamHI site on the 3′ end. Twonon-coding sequences are amplified via PCR: one non-coding sequence(fragment 1) is amplified with a HindIII site at the 5′ end and an Xbasite at the 3′ end; the other non-coding sequence (fragment 2) isamplified with a BamHI site at the 5′ end and a HindIII site at the 3′end. The CMV promoter and the fragments (1 and 2) are digested with theappropriate enzymes (CMV promoter—XbaI and BamHI; fragment 1—XbaI;fragment 2—BamHI) and ligated together. The resulting ligation productis digested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5.×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37 degree C. The following day, the media isaspirated and replaced with 10 ml of fresh media and incubated for afurther 16-24 hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 28 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat disorders, diseases and conditions. The gene therapymethod relates to the introduction of naked nucleic acid (DNA, RNA, andantisense DNA or RNA) sequences into an animal to increase or decreasethe expression of the polypeptide. The polynucleotide of the presentinvention may be operatively linked to a promoter or any other geneticelements necessary for the expression of the polypeptide by the targettissue. Such gene therapy and delivery techniques and methods are knownin the art, see, for example, WO90/11092, WO98/11779; U.S. Pat. Nos.5,693,622, 5,705,151, 5,580,859; Tabata et al., Cardiovasc. Res.35(3):470-479 (1997); Chao et al., Pharmacol. Res. 35(6):517-522 (1997);Wolff, Neuromuscul. Disord. 7(5): 314-318 (1997); Schwartz et al., GeneTher. 3(5):405-411 (1996); Tsurumi et al., Circulation 94(12):3281-3290(1996) (incorporated herein by reference).

The polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The polynucleotide constructs canbe delivered in a pharmaceutically acceptable liquid or aqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the polynucleotides of the present invention may alsobe delivered in liposome formulations (such as those taught in FelgnerP. L. et al. (1995) Ann. N.Y. Acad. Sci. 772:126-139 and Abdallab B. etal. (1995) Biol. Cell 85(1):1-7) which can be prepared by methods wellknown to those skilled in the art.

The polynucleotide vector constructs used in the gene therapy method arepreferably constructs that will not integrate into the host genome norwill they contain sequences that allow for replication. Any strongpromoter known to those skilled in the art can be used for driving theexpression of DNA. Unlike other gene therapies techniques, one majoradvantage of introducing naked nucleic acid sequences into target cellsis the transitory nature of the polynucleotide synthesis in the cells.Studies have shown that non-replicating DNA sequences can be introducedinto cells to provide production of the desired polypeptide for periodsof up to six months.

The polynucleotide construct can be delivered to the interstitial spaceof tissues within the an animal, including of muscle, skin, brain, lung,liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked polynucleotide injection, an effective dosage amount ofDNA or RNA will be in the range of from about 0.05 g/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, nakedpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected polynucleotide in muscle in vivois determined as follows. Suitable template DNA for production of mRNAcoding for polypeptide of the present invention is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The template DNA is injected in 0.1 ml of carrier in a 1 ccsyringe through a 27 gauge needle over one minute, approximately 0.5 cmfrom the distal insertion site of the muscle into the knee and about 0.2cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for protein expression. A time course for protein expression maybe done in a similar fashion except that quadriceps from different miceare harvested at different times. Persistence of DNA in muscle followinginjection may be determined by Southern blot analysis after preparingtotal cellular DNA and HIRT supernatants from injected and control mice.The results of the above experimentation in mice can be use toextrapolate proper dosages and other treatment parameters in humans andother animals using naked DNA.

Example 29 Transgenic Animals

The polypeptides of the invention can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep,cows and non-human primates, e.g., baboons, monkeys, and chimpanzees maybe used to generate transgenic animals. In a specific embodiment,techniques described herein or otherwise known in the art, are used toexpress polypeptides of the invention in humans, as part of a genetherapy protocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g, head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of polypeptides of the present invention, studying conditionsand/or disorders associated with aberrant expression, and in screeningfor compounds effective in ameliorating such conditions and/ordisorders.

Example 30 Knock-Out Animals

Endogenous gene expression can also be reduced by inactivating or“knocking out” the gene and/or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., Nature 317:230-234 (1985);Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g. by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe polypeptides of the invention. The engineered cells which expressand preferably secrete the polypeptides of the invention can beintroduced into the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Transgenic and “knock-out” animals of the invention have uses whichinclude, but are not limited to, animal model systems useful inelaborating the biological function of polypeptides of the presentinvention, studying conditions and/or disorders associated with aberrantexpression, and in screening for compounds effective in amelioratingsuch conditions and/or disorders.

Example 31 Isolation of Antibody Fragments Directed Against Polypeptidesof the Invention from a Library of scFvs

Naturally occurring V-genes isolated from human PBLs are constructedinto a large library of antibody fragments which contain reactivitiesagainst a polypeptide having the amino acid sequence of SEQ ID NO:Y towhich the donor may or may not have been exposed (see e.g., U.S. Pat.No. 5,885,793 incorporated herein in its entirety by reference).

Rescue of the Library.

A library of scFvs is constructed from the RNA of human PBLs asdescribed in WO92/01047. To rescue phage displaying antibody fragments,approximately 10⁹ E. coli harboring the phagemid are used to inoculate50 ml of 2× TY containing 1% glucose and 100 micrograms/ml of ampicillin(2× TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking. Five ml ofthis culture is used to inoculate 50 ml of 2× TY-AMP-GLU, 2×108 TU ofdelta gene 3 helper (M13 delta gene III, see WO92/01047) are added andthe culture incubated at 37° C. for 45 minutes without shaking and thenat 37° C. for 45 minutes with shaking. The culture is centrifuged at4000 r.p.m. for 10 min. and the pellet resuspended in 2 liters of of 2×TY containing 100 micrograms/ml ampicillin and 50 micrograms/mlkanamycin and grown overnight Phage are prepared as described inWO92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harboring a pUC19 derivative supplying the wild type geneIII protein during phage morphogenesis. The culture is incubated for 1hour at 37° C. without shaking and then for a further hour at 37° C.with shaking. Cells were spun down (IEC-Centra 8, 4000 revs/min for 10min), resuspended in 300 ml 2× TY both containing 100 microgramsampicillin/ml and 25 micrograms kanamycin/ml (2× TY-AMP-KAN) and grownovernight, shaking at 37° C. Phage particles are purified andconcentrated from the culture medium by two PEG-precipitations (Sambrooket al., 1990), resuspended in 2 ml PBS and passed through a 0.45micrometer filter (Minisart NML; Sartorius) to give a finalconcentration of approximately 10¹³ transducing units/ml(ampicillin-resistant clones).

Panning the Library.

Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100micrograms/ml or 10 micrograms/ml of a polypeptide of the presentinvention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37° C.and then washed 3 times in PBS. Approximately 10¹³ TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100micrograms/ml ampicillin. The resulting bacterial library is thenrescued with delta gene 3 helper phage as described above to preparephage for a subsequent round of selection. This process is then repeatedfor a total of 4 rounds of affinity purification with tube-washingincreased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS forrounds 3 and 4.

Characterization of Binders.

Eluted phage from the third and fourth rounds of selection are used toinfect E. coli HB 2151 and soluble scFv is produced (Marks, et al.,1991) from single colonies for assay. ELISAs are performed withmicrotiter plates coated with either 10 picograms/ml of the polypeptideof the present invention in 50 mM bicarbonate pH 9.6. Clones positive inELISA are further characterized by PCR fingerprinting (see e.g.,WO92/01047) and then by sequencing.

Example 32 Assays Detecting Stimulation or Inhibition of B cellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL-10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Assays which allow for thedetection and/or observation of the proliferation and differentiation ofthese B-cell populations and their precursors are valuable tools indetermining the effects various proteins may have on these B-cellpopulations in terms of proliferation and differentiation. Listed beloware two assays designed to allow for the detection of thedifferentiation, proliferation, or inhibition of B-cell populations andtheir precursors.

In Vitro Assay—Purified polypeptides of the invention, or truncatedforms thereof, is assessed for its ability to induce activation,proliferation, differentiation or inhibition and/or death in B-cellpopulations and their precursors. The activity of the polypeptides ofthe invention on purified human tonsillar B cells, measuredqualitatively over the dose range from 0.1 to 10,000 ng/mL, is assessedin a standard B-lymphocyte co-stimulation assay in which purifiedtonsillar B cells are cultured in the presence of either formalin-fixedStaphylococcus aureus Cowan I (SAC) or immobilized anti-human IgMantibody as the priming agent. Second signals such as IL-2 and IL-15synergize with SAC and IgM crosslinking to elicit B cell proliferationas measured by tritiated-thymidine incorporation. Novel synergizingagents can be readily identified using this assay. The assay involvesisolating human tonsillar B cells by magnetic bead (MACS) depletion ofCD3-positive cells. The resulting cell population is greater than 95% Bcells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁵ B-cells suspended in culture medium(RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2 ME, 100 U/ml penicillin, 10ug/ml steptomycin, and 10⁻⁵ dilution of SAC) in a total volume of 150ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well)) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of a polypeptide of the invention, or truncated formsthereof Mice receive this treatment for 4 consecutive days, at whichtime they are sacrificed and various tissues and serum collected foranalyses. Comparison of H&E sections from normal spleens and spleenstreated with polypeptides of the invention identify the results of theactivity of the polypeptides on spleen cells, such as the diffusion ofperiarterial lymphatic sheaths, and/or significant increases in thenucleated cellularity of the red pulp regions, which may indicate theactivation of the differentiation and proliferation of B-cellpopulations. Immunohistochemical studies using a B cell marker,anti-CD45R(B220), are used to determine whether any physiologicalchanges to splenic cells, such as splenic disorganization, are due toincreased B-cell representation within loosely defined B-cell zones thatinfiltrate established T-cell regions.

Flow cytometric analyses of the spleens from mice treated withpolypeptide is used to indicate whether the polypeptide specificallyincreases the proportion of ThB+, CD45R(B220)dull B cells over thatwhich is observed in control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andpolypeptide-treated mice.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides of theinvention (e.g., gene therapy), agonists, and/or antagonists ofpolynucleotides or polypeptides of the invention.

Example 33 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4degrees C. (1 μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washedthree times with PBS, PBMC are isolated by F/H gradient centrifugationfrom human peripheral blood and added to quadruplicate wells(5×10⁴/well) of mAb coated plates in RPMI containing 10% FCS and P/S inthe presence of varying concentrations of polypeptides of the invention(total volume 200 ul). Relevant protein buffer and medium alone arecontrols. After 48 hr. culture at 37 degrees C., plates are spun for 2min. at 1000 rpm and 100 μl of supernatant is removed and stored −20degrees C. for measurement of IL-2 (or other cytokines) if effect onproliferation is observed. Wells are supplemented with 100 ul of mediumcontaining 0.5 uCi of ³H-thymidine and cultured at 37 degrees C. for18-24 hr. Wells are harvested and incorporation of ³H-thymidine used asa measure of proliferation. Anti-CD3 alone is the positive control forproliferation. IL-2 (100 U/ml) is also used as a control which enhancesproliferation. Control antibody which does not induce proliferation of Tcells is used as the negative controls for the effects of polypeptidesof the invention.

The studies described in this example tested activity of polypeptides ofthe invention. However, one skilled in the art could easily modify theexemplified studies to test the activity of polynucleotides of theinvention (e.g., gene therapy), agonists, and/or antagonists ofpolynucleotides or polypeptides of the invention.

Example 34 Effect of Polypeptides of the Invention on the Expression ofMHC Class II, Costimulatory and Adhesion Molecules and CellDifferentiation of Monocytes and Monocyte—Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-α,causes a rapid change in surface phenotype (increased expression of MRCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of polypeptides of theinvention or LPS (positive control), washed with PBS containing 1% BSAand 0.02 mM sodium azide, and then incubated with 1:20 dilution ofappropriate FITC— or PE-labeled monoclonal antibodies for 30 minutes at4 degrees C. After an additional wash, the labeled cells are analyzed byflow cytometry on a FACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (10⁶/ml) are treated with increasing concentrations ofpolypeptides of the invention for 24 hours. LPS (100 ng/ml) is added tothe cell culture as positive control. Supernatants from the cellcultures are then collected and analyzed for IL-12 content usingcommercial ELISA kit (e.g, R & D Systems (Minneapolis, Minn.)). Thestandard protocols provided with the kits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fe receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations ofpolypeptides of the invention or LPS (positive control), washed with PBScontaining 1% BSA and 0.02 mM sodium azide, and then incubated with 1:20dilution of appropriate FITC- or PE-labeled monoclonal antibodies for 30minutes at 4 degrees C. After an additional wash, the labeled cells areanalyzed by flow cytometry on a FACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. Polypeptides, agonists, or antagonists of the invention canbe screened using the three assays described below. For each of theseassays, Peripheral blood mononuclear cells (PBMC) are purified fromsingle donor leukopacks (American Red Cross, Baltimore, Md.) bycentrifugation through a Histopaque gradient (Sigma). Monocytes areisolated from PBMC by counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated process (apoptosis). Addition tothe culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the compoundto be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBScontaining PI at a final concentration of 5 μg/ml, and then incubated atroom temperature for 5 minutes before FACScan analysis. PI uptake hasbeen demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of the a polypeptide of the invention andunder the same conditions, but in the absence of the polypeptide. ForIL-12 production, the cells are primed overnight with IFN (100 U/ml) inpresence of a polypeptide of the invention. LPS (10 ng/ml) is thenadded. Conditioned media are collected after 24 h and kept frozen untiluse. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is then performedusing a commercially available ELISA kit (e.g, R & D Systems(Minneapolis, Minn.)) and applying the standard protocols provided withthe kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×10⁵cell/well. Increasing concentrations of polypeptides of the inventionare added to the wells in a total volume of 0.2 ml culture medium (RPMI1640+10% FCS, glutamine and antibodies) After 3 days incubation, theplates are centrifuged and the medium is removed from the wells. To themacrophage monolayers, 0.2 ml per well of phenol red solution (140 mMNaCl, 10 mM potassium phosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mMphenol red and 19 U/ml of HRPO) is added, together with the stimulant(200 nM PMA). The plates are incubated at 37° C. for 2 hours and thereaction is stopped by adding 20 μl 1N NaOH per well. The absorbance isread at 610 nm. To calculate the amount of H₂O₂ produced by themacrophages, a standard curve of a H₂O₂ solution of known molarity isperformed for each experiment.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polypeptides,polynucleotides (e.g., gene therapy), agonists, and/or antagonists ofthe invention.

Example 35 Biological Effects of Polypeptides of the Invention

Astrocyte and Neuronal Assays

Recombinant polypeptides of the invention, expressed in Escherichia coliand purified as described above, can be tested for activity in promotingthe survival, neurite outgrowth, or phenotypic differentiation ofcortical neuronal cells and for inducing the proliferation of glialfibrillary acidic protein immunopositive cells, astrocytes. Theselection of cortical cells for the bioassay is based on the prevalentexpression of FGF-1 and FGF-2 in cortical structures and on thepreviously reported enhancement of cortical neuronal survival resultingfrom FGF-2 treatment. A thymidine incorporation assay, for example, canbe used to elucidate a polypeptide of the invention's activity on thesecells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad Sci USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of a polypeptideof the invention to induce neurite outgrowth can be compared to theresponse achieved with FGF-2 using, for example, a thymidineincorporation assay.

Fibroblast and Endothelial Cell Assays

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells can be cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells are then incubatedfor one day in 0.1% BSA basal medium. After replacing the medium withfresh 0.1% BSA medium, the cells are incubated with the test proteinsfor 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) isadded to each well to a final concentration of 10%. The cells areincubated for 4 hr. Cell viability is measured by reading in a CytoFluorfluorescene reader. For the PGE₂ assays, the human lung fibroblasts arecultured at 5,000 cells/well in a 96-well plate for one day.After amedium change to 0.1% BSA basal medium, the cells are incubated withFGF-2 or polypeptides of the invention with or without IL-1α for 24hours. The supernatants are collected and assayed for PGE₂ by EIA kit(Cayman, Ann Arbor, Mich.). For the IL-6 assays, the human lungfibroblasts are cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells areincubated with FGF-2 or with or without polypeptides of the inventionIL-1a for 24 hours. The supernatants are collected and assayed for IL-6by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or polypeptides of theinvention for 3 days in basal medium before the addition of Alamar Blueto assess effects on growth of the fibroblasts. FGF-2 should show astimulation at 10-2500 ng/ml which can be used to compare stimulationwith polypeptides of the invention.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine(MPP⁺) and released.Subsequently MPP⁺ is actively accumulated in dopaminergic neurons by thehigh-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, polypeptides of the invention can beevaluated to determine whether it has an action similar to that of FGF-2in enhancing dopaminergic neuronal survival in vitro and it can also betested in vivo for protection of dopaminergic neurons in the striatumfrom the damage associated with MPTP treatment. The potential effect ofa polypeptide of the invention is first examined in vitro in adopaminergic neuronal cell culture paradigm. The cultures are preparedby dissecting the midbrain floor plate from gestation day 14 Wistar ratembryos. The tissue is dissociated with trypsin and seeded at a densityof 200,000 cells/cm² on polyorthinine-laminin coated glass coverslips.The cells are maintained in Dulbecco's Modified Eagle's medium and F12medium containing hormonal supplements (N1). The cultures are fixed withparaformaldehyde after 8 days in vitro and are processed for tyrosinehydroxylase, a specific marker for dopminergic neurons,immunohistochemical staining. Dissociated cell cultures are preparedfrom embryonic rats. The culture medium is changed every third day andthe factors are also added at that time.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if a polypeptide of the invention acts to prolong thesurvival of dopaminergic neurons, it would suggest that the polypeptidemay be involved in Parkinson's Disease.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 36 The Effect of Polypeptides of the Invention on the Growth ofVascular Endothelia Cells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. Apolypeptide having the amino acid sequence of SEQ ID NO:Y, and positivecontrols, such as VEGF and basic FGF (bFGF) are added, at varyingconcentrations. On days 4 and 6, the medium is replaced. On day 8, cellnumber is determined with a Coulter Counter.

An increase in the number of HUEC cells indicates that the polypeptideof the invention may proliferate vascular endothelial cells.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 37 Stimulatory Effect of Polypeptides of the Invention on theProliferation of Vascular Endothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF₁₆₅ or a polypeptide of the invention in 0.5%FBS) with or without Heparin (8 U/ml) are added to wells for 48 hours.20 mg of MTS/PMS mixture (1:0:05) are added per well and allowed toincubate for 1 hour at 37° C. before measuring the absorbance at 490 nmin an ELISA plate reader. Background absorbance from control wells (somemedia, no cells) is subtracted, and seven wells are performed inparallel for each condition. See, Leak et al. In Vitro Cell. Dev. Biol.30A:512-518 (1994).

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 38 Inhibition of PDGF—Induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4 degrees C. for 2 h after being exposedto denaturing solution and then incubated with thestreptavidin-peroxidase and diaminobenzidine. After counterstaining withhematoxylin, the cells are mounted for microscopic examination, and theBrdUrd-positive cells are counted. The BrdUrd index is calculated as apercent of the BrdUrd-positive cells to the total cell number. Inaddition, the simultaneous detection of the BrdUrd staining (nucleus)and the FITC uptake (cytoplasm) is performed for individual cells by theconcomitant use of bright field illumination and dark field-UVfluorescent illumination. See, Hayashida et al., J. Biol. Chem.6:271(36):21985-21992 (1996).

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 39 Stimulation of Endothelial Migration

This example will be used to explore the possibility that a polypeptideof the invention may stimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, MD; Falk, W., etal., J. Immunological Methods 1980;33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO2 to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 40 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, activity of apolypeptide of the invention can be assayed by determining nitric oxideproduction by endothelial cells in response to the polypeptide.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andthe polypeptide of the invention. Nitric oxide in the medium isdetermined by use of the Griess reagent to measure total nitrite afterreduction of nitric oxide-derived nitrate by nitrate reductase. Theeffect of the polypeptide of the invention on nitric oxide release isexamined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.) (1049). Calibration of the NO elementsis performed according to the following equation:2 KNO₂+2 KI+2 H₂SO₄6 2NO+I₂+2H₂O+2K₂SO₄

The standard calibration curve is obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing KI and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and HUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×10⁶ endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

The studies described in this example tested activity of polypeptides ofthe invention. However, one skilled in the art could easily modify theexemplified studies to test the activity of polynucleotides (e.g., genetherapy), agonists, and/or antagonists of the invention.

Example 41 Effect of Polypepides of the Invention on Cord Formation inAngiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Chord FormationMedium containing control buffer or a polypeptide of the invention (0.1to 100 ng/ml) and the cells are cultured for an additional 48 hr. Thenumbers and lengths of the capillary-like chords are quantitated throughuse of the Boeckeler VIA-170 video image analyzer. All assays are donein triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 42 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of polypeptides of the invention tostimulate angiogenesis in CAM can be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. Thermanoxcoverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors are dissolved indistilled water and about 3.3 mg/5 ml are pipetted on the disks. Afterair-drying, the inverted disks are applied on CAM. After 3 days, thespecimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsedin 0.12 M sodium cacodylate buffer. They are photographed with a stereomicroscope [Wild M8] and embedded for semi- and ultrathin sectioning asdescribed above. Controls are performed with carrier disks alone.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 43 Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of a polypeptide of the invention measuresthe ability of an existing capillary network to form new vessels in animplanted capsule of murine extracellular matrix material (Matrigel).The protein is mixed with the liquid Matrigel at 4 degree C. and themixture is then injected subcutaneously in mice where it solidifies.After 7 days, the solid “plug” of Matrigel is removed and examined forthe presence of new blood vessels. Matrigel is purchased from BectonDickinson Labware/Collaborative Biomedical Products.

When thawed at 4 degree C. the Matrigel material is a liquid. TheMatrigel is mixed with a polypeptide of the invention at 150 ng/ml at 4degrees C. and drawn into cold 3 ml syringes. Female C57B1/6 miceapproximately 8 weeks old are injected with the mixture of Matrigel andexperimental protein at 2 sites at the midventral aspect of the abdomen(0.5 ml/site). After 7 days, the mice are sacrificed by cervicaldislocation, the Matrigel plugs are removed and cleaned (i.e., allclinging membranes and fibrous tissue is removed). Replicate whole plugsare fixed in neutral buffered 10% formaldehyde, embedded in paraffm andused to produce sections for histological examination after stainingwith Masson's Trichrome. Cross sections from 3 different regions of eachplug are processed. Selected sections are stained for the presence ofvWF. The positive control for this assay is bovine basic FGF (150ng/ml). Matrigel alone is used to determine basal levels ofangiogenesis.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 44 Rescue of Ischemia in Rabbit Lower Limb Model

To study the in vivo effects of polynucleotides and polypeptides of theinvention on ischemia, a rabbit hindlimb ischemia, a rabbit hindlimbischemia model is created by surgical removal of one femoral arteries asdescribed previously (Takeshita et al., Am J. Pathol 147:1649-1660(1995)). The excision of the femoral artery results in retrogradepropagation of thrombus and occlusion of the external iliac artery.Consequently, blood flow to the ischemic limb is dependent uponcollateral vessels originating from the internal iliac artery (Takeshitaet al. Am J. Pathol 147:1649-1660 (1995)). An interval of 10 days isallowed for post-operative recovery of rabbits and development ofendogenous collateral vessels. At 10 day post-operatively (day 0), afterperforming a baseline angiogram, the internal iliac artery of theischemic limb is transfected with 500 mg naked expression plasmidcontaining a polynucleotide of the invention by arterial gene transfertechnology using a hydrogel-coated balloon catheter as described(Riessen et al. Hum Gene Ther. 4:749-758 (1993); Leclerc et al. J. Clin.Invest. 90: 936-944 (1992)). When a polypeptide of the invention is usedin the treatment, a single bolus of 500 mg polypeptide of the inventionor control is delivered into the internal iliac artery of the ischemiclimb over a period of 1 min. through an infusion catheter. On day 30,various parameters are measured in these rabbits: (a) BP ratio—The bloodpressure ratio of systolic pressure of the ischemic limb to that ofnormal limb; (b) Blood Flow and Flow Reserve—Resting FL: the blood flowduring undilated condition and Max FL: the blood flow during fullydilated condition (also an indirect measure of the blood vessel amount)and Flow Reserve is reflected by the ratio of max FL: resting FL; (c)Angiographic Score—This is measured by the angiogram of collateralvessels. A score is determined by the percentage of circles in anoverlaying grid that with crossing opacified arteries divided by thetotal number m the rabbit thigh; (d) Capillary density—The number ofcollateral capillaries determined in light microscopic sections takenfrom hindlimbs.

The studies described in this example tested activity of polynucleotidesand polypeptides of the invention. However, one skilled in the art couldeasily modify the exemplified studies to test the agonists, and/orantagonists of the invention.

Example 45 Effect of Polypeptides of the Invention on Vasodilation

Since dilation of vascular endothelium is important in reducing bloodpressure, the ability of polypeptides of the invention to affect theblood pressure in spontaneously hypertensive rats (SHR) is examined.Increasing doses (0, 10, 30, 100, 300, and 900 mg/kg) of thepolypeptides of the invention are administered to 13-14 week oldspontaneously hypertensive rats (SHR). Data are expressed as themean±SEM. Statistical analysis are performed with a paired t-test andstatistical significance is defined as p<0.05 vs. the response to bufferalone.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 46 Rat Ischemic Skin Flop Model

The evaluation parameters include skin blood flow, skin temperature, andfactor VIII immunohistochemistry or endothelial alkaline phosphatasereaction. Expression of polypeptides of the invention, during the skinischemia, is studied using in situ hybridization.

The study in this model is divided into three parts as follows:

a) Ischemic skin

b) Ischemic skin wounds

c) Normal wounds

The experimental protocol includes:

a) Raising a 3×4 cm, single pedicle full-thickness random skin flap(myocutaneous flap over the lower back of the animal).

b) An excisional wounding (4-6 mm in diameter) in the ischemic skin(skin-flap).

c) Topical treatment with a polypeptide of the invention of theexcisional wounds (day 0, 1, 2, 3, 4 post-wounding) at the followingvarious dosage ranges: 1 mg to 100 mg.

d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21post-wounding for histological, immunohistochemical, and in situstudies.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 47 Peripheral Arterial Disease Model

Angiogenic therapy using a polypeptide of the invention is a noveltherapeutic strategy to obtain restoration of blood flow around theischemia in case of peripheral arterial diseases. The experimentalprotocol includes:

a) One side of the femoral artery is ligated to create ischemic muscleof the hindlimb, the other side of hindlimb serves as a control.

b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, isdelivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 2-3 weeks.

c) The ischemic muscle tissue is collected after ligation of the femoralartery at 1, 2, and 3 weeks for the analysis of expression of apolypeptide of the invention and histology. Biopsy is also performed onthe other side of normal muscle of the contralateral hindlimb.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 48 Ischemic Myocardial Disease Model

A polypeptide of the invention is evaluated as a potent mitogen capableof stimulating the development of collateral vessels, and restructuringnew vessels after coronary artery occlusion. Alteration of expression ofthe polypeptide is investigated in situ. The experimental protocolincludes:

a) The heart is exposed through a left-side thoracotomy in the rat.Immediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

b) a polypeptide of the invention, in a dosage range of 20 mg-500 mg, isdelivered intravenously and/or intramuscularly 3 times (perhaps more)per week for 24 weeks.

c) Thirty days after the surgery, the heart is removed andcross-sectioned for morphometric and in situ analyzes.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 49 Rat Corneal Wound Healing Model

This animal model shows the effect of a polypeptide of the invention onneovascularization. The experimental protocol includes:

a) Making a 1-1.5 mm long incision from the center of cornea into thestromal layer.

b) Inserting a spatula below the lip of the incision facing the outercorner of the eye.

c) Making a pocket (its base is 1-1.5 mm form the edge of the eye).

d) Positioning a pellet, containing 50 ng-5 ug of a polypeptide of theinvention, within the pocket.

e) Treatment with a polypeptide of the invention can also be appliedtopically to the corneal wounds in a dosage range of 20 mg-500 mg (dailytreatment for five days).

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 50 Diabetic Mouse and Glucocordcoid-Impaired Wound HealingModels

A. Diabetic db+/db+ Mouse Model.

To demonstrate that a polypeptide of the invention accelerates thehealing process, the genetically diabetic mouse model of wound healingis used. The full thickness wound healing model in the db+/db+ mouse isa well characterized, clinically relevant and reproducible model ofimpaired wound healing. Healing of the diabetic wound is dependent onformation of granulation tissue and re-epithelialization rather thancontraction (Gartner, M. H. et al, J. Surg. Res. 52:389 (1992);Greenhalgh, D. G. et al., Am. J Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al, Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

A polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) untreated group, and 3)treated group.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffm embedded blocksare sectioned perpendicular to the wound surface (5 mm) and cut using aReichert-Jung microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds are used to assess whether the healing process and themorphologic appearance of the repaired skin is altered by treatment witha polypeptide of the invention. This assessment included verification ofthe presence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometeris used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer can serve as a positive tissuecontrol and human brain tissue can be used as a negative tissue control.Each specimen includes a section with omission of the primary antibodyand substitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

B. Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, Glucocorticoids and Woundhealing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahlet al., J. Immunol. 115: 476-481 (1975);Werb et al., J Exp. Med. 147:1684-1694 (1978)). Glucocorticoids retardwound healing by inhibiting angiogenesis, decreasing vascularpermeability (Ebert et al., An. Intern. Med. 37:701-705 (1952)),fibroblast proliferation, and collagen synthesis (Beck et al., GrowthFactors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797(1978)) and producing a transient reduction of circulating monocytes(Haynes et al., J. Clin. Invest. 61: 703-797 (1978); Wahl,“Glucocorticoids and wound healing”, In: Antiinflammatory SteroidAction: Basic and Clinical Aspects, Academic Press, New York, pp.280-302 (1989)). The systemic administration of steroids to impairedwound healing is a well establish phenomenon in rats (Beck et al, GrowthFactors. 5: 295-304 (1991); Haynes et al., J. Clin. Invest. 61: 703-797(1978); Wahl, “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280-302 (1989); Pierce et al., Proc. Natl. Acad. Sci. USA 86:2229-2233 (1989)).

To demonstrate that a polypeptide of the invention can accelerate thehealing process, the effects of multiple topical applications of thepolypeptide on full thickness excisional skin wounds in rats in whichhealing has been impaired by the systemic administration ofmethylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

The polypeptide of the invention is administered using at a rangedifferent doses, from 4 mg to 500 mg per wound per day for 8 days invehicle. Vehicle control groups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1is 64 mm², the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith a polypeptide of the invention. A calibrated lens micrometer isused by a blinded observer to determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 51 Lymphadema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects of apolypeptide of the invention in lymphangiogenesis and re-establishmentof the lymphatic circulatory system in the rat hind limb. Effectivenessis measured by swelling volume of the affected limb, quantification ofthe amount of lymphatic vasculature, total blood plasma protein, andhistopathology. Acute lymphedema is observed for 7-10 days. Perhaps moreimportantly, the chronic progress of the edema is followed for up to 3-4weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated. The 2 proximal and 2 distal lymphatic vessels and distal bloodsupply of the popliteal node are then and ligated by suturing. Thepopliteal lymph node, and any accompanying adipose tissue, is thenremoved by cutting connective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (AJ Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith Pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software(Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and then at conclusion for total protein andCa2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs are amputated using a quillitine, then bothexperimental and control legs are cut at the ligature and weighed. Asecond weighing is done as the tibio-cacaneal joint is disarticulatedand the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80 EC until sectioning. Upon sectioning, the muscle is observedunder fluorescent microscopy for lymphatics.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 52 Suppression of TNF Alpha-Induced Adhesion Molecule ExpressionBy a Polypeptide of the Invention

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine,is a stimulator of all three CAMs on endothelial cells and may beinvolved in a wide variety of inflammatory responses, often resulting ina pathological outcome.

The potential of a polypeptide of the invention to mediate a suppressionof TNF-a induced CAM expression can be examined. A modified ELISA assaywhich uses ECs as a solid phase absorbent is employed to measure theamount of CAM expression on TNF-a treated ECs when co-stimulated with amember of the FGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC)cultures are obtained from pooled cord harvests and maintained in growthmedium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCSand 1% penicillin/streptomycin in a 37 degree C. humidified incubatorcontaining 5% CO₂. HUVECs are seeded in 96-well plates at concentrationsof 1×10⁴ cells/well in EGM medium at 37 degree C. for 18-24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 supplemented with 100 U/ml penicillinand 100 mg/ml streptomycin, and treated with a given cytokine and/orgrowth factor(s) for 24 h at 37 degree C. Following incubation, thecells are then evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard96 well plate to confluence. Growth medium is removed from the cells andreplaced with 90 ul of 199 Medium (10% FBS). Samples for testing andpositive or negative controls are added to the plate in triplicate (in10 ul volumes). Plates are incubated at 37 degree C. for either 5 h(selectin and integrin expression) or 24 h (integrin expression only).Plates are aspirated to remove medium and 100 μl of 0.1%parafommaldehyde-PBS(with Ca++ and Mg++) is added to each well. Platesare held at 4° C. for 30 min.

Fixative is then removed from the wells and wells are washed 1× withPBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μL of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000dilution) to each well and incubated at 37° C. for 30 min. Wells arewashed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol PhosphatepNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPPsubstrate in glycine buffer is added to each test well. Standard wellsin triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in glycine buffer:1:5,000(10⁰)>10^(−0.5)>10⁻¹>10^(−1.5) 0.5 μl of each dilution is addedto triplicate wells and the resulting AP content in each well is 5.50ng, 1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be addedto each of the standard wells. The plate must be incubated at 37° C. for4 h. A volume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

The studies described in this example tested activity of a polypeptideof the invention. However, one skilled in the art could easily modifythe exemplified studies to test the activity of polynucleotides (e.g.,gene therapy), agonists, and/or antagonists of the invention.

Example 53 Cloning, Sequence Analysis and Chromosomal Localization ofthe Novel Human Integrin Alpha 11 Subunit

The integrins are a large family of cell adhesion molecules consistingof noncovalently associated αβ heterodimers. We have cloned andsequenced a novel human integrin α-subunit cDNA, designated α11. The α11cDNA encodes a protein with a 22 amino acid signal peptide, a large 1120residue extracellular domain that contains an 1-domain of 207 residuesand is linked by a transmembrane domain to a short cytoplasmic domain of24 amino acids. The deduced α11 protein shows the typical structuralfeatures of integrin α-subunits and is similar to a distinct group ofα-subunits from collagen-binding integrins. However, it differs frommost integrin α-chains by an incompletetely preserved cytoplasmic GFFKRmotif. The human ITGA11 gene was located to bands q22.3-23 on chromosome15, and its transcripts were found predominantly in bone, cartilage aswell as in cardiac and skeletal muscle. Expression of the 5.5 kilobaseα11 mRNA was also detectable in ovary and small intestine.

Introduction

All vertebrate cells express members of the integrin family of celladhesion molecules, which mediate cellular adhesion to other cells andextracellular subtratum, cell migration and participate in importantphysiologic processes from signal transduction to cell proliferation anddifferentiation {Hynes, 92; Springer, 92}. Integrins are structurallyhomologous heterodimeric type-I membrane glycoproteins formed by thenoncovalent association of one of eight β-subunits with one of the 17different α-subunits described to date, resulting in at least 22different αβ complexes. Their binding specificities for cellular andextracellular ligands are determined by both subunits and aredynamically regulated in a cell-type-specific mode by the cellularenvironment as well as by the developmental and activation state of thecell {Diamond and Springer, 94}. In integrin α-subunits, theaminoterminal region of the large extracellular domain consists of aseven-fold repeated structure which is predicted to fold into aβ-propeller domain {Corbi et al., 1987; Springer, 1997}. The three orfour C-terminal repeats contain putative divalent cation binding motifsthat are thought to be important for ligand binding and subunitassociation {Diamond and Springer, 94}. The α¹, α², α¹⁰, α^(D), α^(E),α^(L), α^(M) and α^(X)-subunits contain an approximately 200 amino acidI-domain inserted between the second and third repeat that is notpresent in other α-chains {Larson et al., 1989}. Several isolatedI-domains have been shown to independently bind the ligands of theparent integrin heterodimer {Kamata and Takada, 1994; Randi and Hogg,1994}. The α³, α⁵⁻⁸, α^(IIb) and α^(V) -subunits are proteolyticallyprocessed at a conserved site into disulphide-linked heavy and lightchains, while the α⁴-subunit is cleaved at a more aminoterminal siteinto two fragments that remain noncovalently associated {Hemler et al.,90}. Additional α-subunit variants are generated by alternative splicingof primary transcripts {Ziober et al., 93; Delwel et al., 95; Leung etal., 98}. The extracellular domains of α-integrin subunits are connectedby a single spanning transmembrane domain to short, diverse cytoplasmicdomains whose only conserved feature is a membrane-proximal KXGFF(K/R)Rmotif {Sastry and Horwitz, 1993}. The cytoplasmic domains have beenimplicated in the cell-type-specific modulation of integrin affinitystates {Williams et al., 1994}.

Here we report the cDNA cloning, sequence analysis, expression andchromosomal localization of the human α-integrin subunit.

Materials and Methods

Library Screening and DNA Sequencing.

A human fetal heart cDNA library in λgt10 (Clontech Laboratories, Inc.,Palo Alto, Calif., USA) was screened with ³²P-labelled (rediprime,Amersham New Zealand Ltd., Auckland, New Zealand) probes correspondingto the regions 473 to 749 and 2394 to 3189 of the α11 cDNA usingstandard procedures. Inserts were subcloned from λgt10 into pUC21 andsequenced on both strands according to a successive specific primerstrategy on an automated sequencer (Applied Biosystems 373A, The Centrefor Gene Technology, School of Biological Sciences, The University ofAuckland).

Northern Blot Analysis and Tissue Distribution.

A 1341 bp PCR fragment corresponding to the region 351-1692 of the αcDNA was ³²P-labelled (rediprime) and hybridized with human multipletissue Northern blots (MTN I and MTN II, Clontech) for 16 h at 60∞C inExpressHyb solution (Clontech). Filters were washed twice with0.1×SSC/1% SDS at 50∞C for 30 min, and autoradiographed. Human DNA from63 tissue-specific cDNA libraries (Express-Check™, American Type CultureCollection, Manassas, Va., USA) was amplified using primers KL120(5′-GCAGGGATGCCACCTGCC) and KL119 (5′-GATGAAGACTGTGGTGTCGAAGG) accordingto the manufacturers instructions. PCR-products were resolved by agarosegel electrophoresis and transferred to Hybond C+ (Amersham). Filterswere hybridized by standard procedures {Ausubel et al., 98} with a 502bp ³²P-labelled (rediprime) probe fragment obtained from the cloned ″¹¹cDNA with the same oligonucleotides.

Chromosomal Assignment.

500 ng genomic DNA prepared from a panel of 21 human-rodent somatic cellhybrids or from human, mouse and hamster cells {Kelsell et al., 95} wasamplified with oligonuleotides KL175 (5′-GGTGCCAGACCTACATGGAC) and KL189(5′-CGTGCAAATTCAATGCCAAATGCC) in a standard PCR reaction of 30 cycles(94∞C for 1 min, 55∞C for 1 min, 72∞C for 2 min). All PCR reactions wereresolved in a 2% agarose gel. Southern hybridization was performed asdetailed above, except that the probe fragment was obtained from cloneHOHBY69 with oligonucleotides KL175 and KL189. For fluorescent in situhybridization, metaphase spreads were prepared fromphytohemagglutinin-stimulated peripheral blood lymphocytes of a 46,XYmale donor using standard cytogenetic procedures. A purified 3.7 kBfragment representing the entire coding region of clone HOHBY69 waslabelled with biotin-16-dUTP using the High Prime labelling kit (RocheMolecular Biochemicals, Auckland, NZ). Conditions for hybridization andimmunofluorescent detection were essentially as described {Morris etal., 93}, except that C₀t-1 suppression was not required, slides werewashed to a stringency of 0,1×SSC/60∞C after hybridization, and anadditional amplification step was needed because of the small size ofthe probe. For precise chromosome band localization, DAPI and FITCimages were captured using a Photometrics KAF1400 CCD camera and QUIPSSmartcapture FISH software version 1.3 (Vysis Inc., Downers Grove, Ill.,USA). QUIPS CGH/Karyotyping software (version 3.0.2) assisted karyotypeanalysis.

Results

Cloning of a Novel Human α-Integrin Subunit cDNA:

A protein homology search {Altschul et al., 90} of the human expressedsequence tag (EST) databases of Human Genome Sciences, Inc. {Ni et al.,97} and The Institute for Genomic Research {Kirkness and Kerlavage, 97}identified the clones HRDAF83 and HOEAM34 as candidate novel integrinα-subunit cDNAs. Clone HRDAF83 was isolated from a humanrhabdomyosarcoma cDNA library and sequenced on both strands. The 1223 bpinsert contains largely incompletely processed hnRNA and a 277 bp regionthat showed homology to the aminoterminal half of the α1-integrin1-domain. The 2517 bp insert of clone HOEAM34 was derived from a humanosteoblast cDNA library. It is homologous to the C-terminal part of thehuman α1-subunit and contains 1324 nucleotides of 3′-untranslatedregion. In order to isolate the full-length cDNAs for these integrinα-subunits, a cDNA library prepared from human fetal heart in λgt10 wasscreened with the 277 bp fragment from clone HRDAF83 homologous to theα1-I-domain. Two clones, λ831 and λ832, were isolated and both strandsof their inserts sequenced. Clone λ832 contains the entire 5′ half of anovel α-subunit cDNA, while clone λ831 covers the same region, but is358 bp and 173 bp shorter than λ831 at its 5′-and 3′-ends, respectively.A screening of the same library with a 795 bp fragment from the extreme5′-terminus of clone HOEAM34 identified clone λ342, which containedessentially the same region as clone HOEAM34 but has a 317 bp shorter3′-untranslated region. Rescreening the EST databases with the sequencesderived from the human fetal heart library led to the identification ofclone HOHBY69, which was isolated from a osteoblast cDNA libray. Bothstrands of the 4681 bp insert of clone HOHBY69 were sequenced. The5′-region of HOHBY69 was identical to the HRDAF83/λ832/λ831-group, whilethe 3′-region of HOHBY69 was largely identical to HOEAM34 and λ342,thereby demonstrating that the two groups of partial cDNAs represent the5′-and 3′-portions of the same cDNA. One major difference between theHOHBY69 and HOEAM34/λ342 is the presence of an additional GTA-triplet atposition 3088 in HOHBY69. From the overlapping clones, a total of 4986bp of cDNA was assembled to the composite sequence shown in FIG. 19A-Fand has been submitted to GenBank™ with accession number AF109681. ThiscDNA encodes a previously unidentifed human integrin α-subunit that wasdesignated α11.

Structure of the Human α11-subunit.

The α11 cDNA contains a 5′-untranslated region of 72 nucleotides and asingle open reading frame extending from a predicted translationinitiation codon at position +1 to a TGA termination codon at position3570. This is followed by 1324 nucleotides of 3′ untranslated regionwhich contains an AATTAAA polyadenylation signal {Wahle and Keller,1996} 12 nucleotides upstream of a poly(A) stretch. The deduced aminoacid sequence contains a 22 residue N-terminal region with thecharacteristics of a cleaved signal peptide {von Heijne, 83; Nielsen etal, 97}, a large extracellular domain of 1120 amino acids followed by a23 amino acid hydrophobic stretch that resembles a transmembrane domain,and a short 24 residue cytoplasmic domain. The molecular weight of themature 1167 amino acid α11-subunit is predicted to be 131 kDa, but theaddition of carbohydrate side chains to any of the 15 potentialN-glycosylation sequons [NX(S/T)] within the extracellular domain islikely to increase the molecular weight of the native protein. AnI-domain of 207 amino acids is inserted between the second and thirdrepeat. Consistent with the structure of an typical I-domain-containingintegrin α-subunit, it lacks a potential dibasic protease cleavage sitein the C-terminal region of the extracellular domain.

The α11-subunit is most closely related to the recently discoveredα10-subunit (Camper et al, 98, Lehnert et al., in preparation} and theα11-and α2-subunits. Overall, the mature α11-protein is 45% identical tothe α10 chain, while the homologies to the α1and α2-subunits are 41% and39%, respectively. Even greater homology exists between the I-domains ofthe α10- and α11-subunits which are 60% identical to each other. Thehigh degree of homology seen in the extracellular domains of thesubunits is in contrast to the low similarity of their cytoplasmicdomains. Interestingly, the KXGFF(K/R)R motif that is absolutelyconserved in all other a-subunit cytoplasmic domains is only partiallypreserved in both subunits. The sequence in α11 is KLGFFRS, while theα10-subunit contains a KLGFFAH motif. A graphical comparison of thesimilarity between all integrin α-subunits is shown in FIG. 3. Togetherwith the α-subunits from the collagen-binding integrins α1β1, α2β1 andα10β1, the α11-subunit forms a group distinct from the otherI-domain-containing integrin subunits.

Tissue Distribution and Expression of the Integrin α11-Subunit.

The tissue distribution of the α11 mRNA was assessed by screeningmultiple human tissue Northern blots with a probe corresponding to theregion 351-1692 of the α11 cDNA. A single transcript of approximately5.5 kb was found weakly expressed only in ovary and small intestine.Integrin α11-subunit expression was further analyzed by amplificationand Southern hybridization of a 502 bp fragment corresponding to theregion 1988-2490 in the all cDNA from tissue-specific human cDNAlibraries. α11 cDNA was detected in five different cDNA librariesprepared from fetal heart (day 57-75), in two fetal brain libraries, andin a cDNA library from large intestine (not shown). An analysis of theHuman Genome Sciences Database revealed eight different α11-related ESTsin human osteoblast libraries, three EST in a human chondrosarcoma cDNAlibrary and two EST in a human stromal osteoclastoma library.

Chromosomal Localization of the Integrin α11-Subunit.

Genomic DNA from a collection af 21 human-rodent somatic cell hybrids{Kelsell et al, 95} was amplified by PCR using oligonucleotide primersdirected the region 473 to 749 of the human α11 cDNA. In Southernhybridization, a signal corresponding to a 1,4 kb fragment wasdetectable only with DNA from a hybrid cell line that contains humanchromosome 15. A fragment of the same size was also amplified from humangenomic DNA, but not from mouse or hamster DNA (FIG. 5C). Cloning andsequence of the PCR product from chromosome 15 revealed the presence ofa 1154 bp intron inserted after cDNA-position 600, thus resulting in aPCR-product of 1431 bp. The ITGA11 gene was also localized byfluorescent in situ hybridization of metaphase chromosomes with theentire coding region from clone HOHBY69. All of 20 metaphase cellsanalyzed showed fluorescent signal on both chromosomes 15, specificallyacross bands q22.3-q23. No additional signals were detected on any otherchromosome (FIG. 5A).

Discussion:

We have cloned and sequenced a novel cDNA encoding a protein that sharesextensive structural homology with integrin α-chains. The aminoterminal22 amino acids of the deduced protein sequence show the characteristicfeatures of a hydrophobic leader peptide, including a signal peptidaserecognition motif at positions −3 and −1 {von Heijne 83}. Proteolyticcleavage of the precursor protein at this position would result in anaminoterminal sequence for the mature α11-chain of FNMD, which issimilar to the consensus sequence[(F/Y)N(L/V)D] of all other integrinα11-subunits {Tuckwell et at, 94}. The N— terminal half of the largeextracellular region of α11 is composed of seven repeats that eachcontain FG-GAP-GxxY consensus motifs (FG-GAP repeats). These repeats canbe found in all integrin al-subunits and are predicted to fold into aβ-propeller domain {Springer, 97}. Inserted between the second and thirdFG-GAP repeats is a 207 amino acid I-domain spanning from glutamine¹³⁸to methionine³⁴⁴. It contains a divalent cation coordination motif thathas been shown to directly bind Mg²⁺ ions in the α^(M) subunit{Michishita et al, 96}. The noncontiguous amino acid side chainsinvolved in the coordination of magnesium or manganese ions have beenidentified by mutagenesis analysis and from crystal structures of theisolated α², α^(L) and α^(M)-subunit I-domains {Emsley et al, 97; Qu andLeahy, 95; Lee et al, 95;}. All residues required for the coordinationof the divalent cations in these subunits are preserved in theα11-I-domain. These are the asparagines at positions 148 and 249, theserine residues at position 150 and 152, and the threonine at position218. The crystal sctructure of the α2-subunit has revealed a small“helix that is not present in the I-domains of the β2-associatedα-subunits. Together with the MIDAS sphere, amino acid residues fromthis C-helix and the adjacent turn region have been proposed to makephysical contacts to a collagen triple helix {Emsley et al, 97}.Interestingly, the small C-helix is structurally conserved in theα-subunits of the collagen-binding integrins α1β1, α2β1 and α10β1, andis also present in the α11 I-domain (G²⁷⁹YYNR²⁸³). In addition,asparagine¹⁵⁴ and histidine²⁵⁸ of the α2-I-domain were predicted tocontact the collagen triple helix, and both are preserved in the α1, α10and α11-I-domains, but not in other integrin α11-subunits. Theconservation of structural motifs required for collagen binding suggeststhat collagen may be a ligand for the α11 integrin. Each of the repeats5-7 of the α11-subunit accomodates the sequence Dx(D/N)xDxxxD. Three orfour copies of these putative divalent cation binding sites areconserved in all integrin α-subunits and their presence is consistentwith the divalent cation requirement for the adhesive function ofintegrins {Larson et al, 89; Fujimura and Phillips, 83; Hynes, 92}. Theextracellular domain of the integrin α11-subunit contains 20 cysteineresidues. Only the intramolecular disulfide bonds in the “^(IIb)subunithave been biochemically characterized {Calvete et al., 89}, but thelocation of many cysteines is conserved in integrin α-subunits. In theα11-subunit, the cysteine residues 637 and 646, 652 and 707, 759 and765, and 859 and 871 are homologous to the residues that form the fourcarboxyterminal disulfide bonds in the heavy chain of”^(IIb) {Calvete etal, 89}. Based on the proposed structure of the integrin α-subunitpropeller domain {Springer et al, 97}, additional disulfide bonds withinthe α11 subunit can be predicted between cysteine residues 54 and 61, 99and 117, and between 107 and 137. Two additional cysteine residues arefound within a short segment (residues 783 to 798) that is unique to theα11-subunit. The integrin cytoplasmic domains play central roles inintegrin affinity modulation and in cellular signal transmission. Themembrane-proximal sequence KxGFF(K/R)R is strictly conserved amongintegrin α-subunit cytoplasmic domains {Williams et al., 94}. Withinthis motif, both phenylalanine residues and the last arginine have beenimplicated in maintaining the default low affinity state of integrinsα^(L)β₂ and α^(IIb)β₃, as their substitution or deletion resulted inconstitutively activated ligand binding {O'Toole et al, 94, Lu andSpriner 97}. Interestingly, the last arginine residue is replaced by aserine in the α11 cytoplasmic domain and with a histidine in the α10subunit, suggesting that both integrins might be in a default “high”affinity state. It will be interesting to analyze whether substitutionof these residues with a conserved arginine will affect their affinitystatus. We have isolated α11 cDNAs from osteoclast, osteoblast,myosarcoma and fetal heart libraries. Amongst the HGS EST databases,integrin α11 transcripts were predominantly found in libraries preparedfrom osteoblast, osteoclast and chondrosarcoma cells. A search forfurther “¹¹-related sequences in the EST division of the GenBankdatabase revealed two clones (accession numbers Z50157 and Z50167) fromprimary human myoblasts {Genini et al, 96}, two clones from humantrabecular bone cells (AA852614 and AA852615), as well as clones fromfibroblast cells (W45078), pancreatic tumor (U53091) and breast tissue(H16112). In contrast, Northern blot analysis detected α11-expressiononly in ovary and small intestine only fetal heart, fetal brain andlarge intestine. Of the tissues represented in the tissue-specificcDNA-library panel, only fetal heart, fetal brain and large intestineshowed detectable α11-expression. However, bone- and muscle-derivedtissues were not included in the Northern blot, and cDNA librariesprepared from these tissues were also not represented in thetissue-specific cDNA panel.

The ITGA11 gene was localized to chromosome 15, bands q22.3-23, by FISHand PCR analysis of human-rodent somatic cell hybrids. This segment isoverrepresented in squamous cell carcinomas {Wolff et al., 1998}, butappears to only infrequently affected in other cancers. Genes at thisregion encode neogenin, a protein expressed ubiquitously expressed inhuman tissues {Meyerhardt et al., 97}; tropomyosin 1, expressed incardiac and skeletal muscle tissues {Tiso et al, 97}; and the humanhomologue of the metalloprotease-disintegrin kuzbanian, which isoverexpressed in tumors of sympathoadrenal origin {Yavari et al, 98}. Inaddition, the region 15q22.3-q23 is linked to Bardet-Biedl syndrome 4, aheterogeneous autosomal disorder characterized by obesity and associatedwith cardiovascular anomalities {Carmi et al., 95}.

In conclusion, we have cloned and sequenced the cDNA for the novelintegrin α11-subunit which is closely related to the “-subunits of thecollagen-binding integrins α1β1, α2β1 and α10β1. The high degree ofhomology of α11 to these subunits suggests that it associates with theintegrin β1-subunit, and may function as an additional collagenreceptor.

All references referred to above and presented below are herebyincorporated herein by reference:

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Example 54 Identification and Characterization of a New Member of theSiglec Family of Sialic Acid Binding Immunoglobulin-Like Lectins

Summary:

Characterized herein is CD33-likeSV, a new member of the Siglec familyof sialic acid binding receptors of the immunoglobulin superfamily. Afull length cDNA encoding CD33-likeSV was isolated from a human spleencDNA library. CD33-likeSV is predicted to contain five extracellularimmunoglobulin-like domains, a transmembrane region and a cytoplasmictail containing three putative tyrosine-based signaling motifs. Overall,CD33-likeSV exhibited a high degree of sequence similarity to genesencoding CD33/Siglec-3, Siglecs-5, -6, -7, -8 and -9 and mapped to thesame region, on chromosome 19q13.3. Phylogenetic analysis and sequencecomparisons indicated that CD33-likeSV was the most ancient of theCD33-related subgroup and may have given rise to the other CD33-relatedSiglecs by a process involving exon deletion and gene duplication. WhenCD33-likeSV was expressed on COS or Chinese hamster ovary cells, it wasable to mediate sialic acid dependent binding to human erythrocytes andsoluble sialoglycoconjugates. Using flow cytometry of human peripheralblood leukocytes, CD33-likeSV was found to be expressed on eosinophils,monocytes and B cells. Higher levels of CD33-likeSV were found on aminor subset of cells within the lymphocyte gate that expressed the CD16low affinity Fc receptor but lacked the CD56 antigen.

Introduction:

Siglecs are sialic acid binding members of the Ig superfamily expressedat the cell surface (1). Structurally, they have a characteristicN-terminal V-set Ig-like domain containing the sialic acid binding site(2) followed by varying numbers of C2 set domains. Most Siglecs have oneor more immune receptor tyrosine-based inhibitory motifs (ITIMs) intheir cytoplasmic tails implicated in negative regulatory signalingfunctions (3-9). The first Siglecs to be defined weresialoadhesin/Siglec-1, CD22/Siglec-2, CD33/Siglec-3 and myelinassociated glycoprotein/Siglec-4 expressed by macrophages, B cells,myeloid cells and myelin forming cells respectively (10-14). Recentstudies have revealed the existence of a new subset of human Siglecsthat are highly related to CD33/Siglec-3, namely Siglecs-5, -6, -7, -8and -9 (6, 15-22). Each protein exhibits sialic acid binding propertiesand is expressed in a characteristic manner. With the exception ofSiglec-6, which is found mostly in the placenta (16), the CD33-relatedgroup of Siglecs are largely expressed on discrete subsets ofhemopoietic cells. Interestingly, the highest levels of expression ofthese proteins are found on effector cells of the innate immune system,including monocytes (Siglecs-3, -5, -7 and -9), neutrophils (Siglecs-5and -9), eosinophils (Siglec-8) and natural killer cells (Siglec-7).This observation, together with the common theme of sialic acidrecognition and the presence of conserved, putative tyrosine-basedinhibitory signaling motifs (4-6, 9), has led to the suggestion thatthese proteins might be involved in regulating activation of leukocytesvia sialic acid recognition (21, 22).

Described herein are the properties of a new member of the Siglecfamily. This protein has 5 extracellular Ig-like domains and based onphylogenetic comparisons it may represent a precursor of theCD33-related Siglec subgroup. Amongst peripheral blood leukocytes,CD33-likeSV was found to be expressed on eosinophils, monocytes and Bcells. Higher levels of this novel Siglec were found on a minor subsetof natural killer-like cells characterized by high levels of the CD16low affinity Fe receptor and an absence of the CD56 antigen.

Experimental Procedures:

Materials

Unless specified otherwise, all reagents and chemicals were purchasedfrom Sigma. ¹²⁵I-streptavidin (20-40 mCi/mg) and protein A-Sepharosewere obtained from Amersham Pharmacia Biotech. Vibrio cholerae sialidasewas purchased from Calbiochem. Biotinylated polyacrylamide (PAA)glycoconjugates carrying either NeuAcα2,3Galβ 1,4Glc (2,3-PAA) orNeuAcα2,6Galβ 1,4Glc (2,6-PAA), were obtained from Syntesome (Munich,Germany). These conjugates have a molecular mass of approximately 30 kDaand contain 20% mol of saccharide and 5% mol of biotin. Phycoerythrin(PE)-conjugated mAbs against the following human CD antigens werepurchased from Serotec (Kidlington, UK): CD3, CD4, CD8, CD16, CD19,CD56. FITC-conjugated F(ab)2 anti-mouse IgG was from Dako, (Cambridge,UK).

Identification and Characterization of CD33-likeSV cDNA

Using the amino acid sequence of CD33, a specific homology search wasperformed against Human Genome Science's database, containing more thanone million expressed sequence tags obtained from over 700 differentcDNA libraries. 21 clones encoding a potential novel Siglec wereidentified in cDNA libraries prepared from the following human sources:bone marrow, unstimulated B cells, eosinophils, primary dendritic cells,spleen, chronic lymphocytic leukemia. Inserts from four clones weresequenced: pHEOMH10 (eosinophil), pHEOOV77 (eosinophil), pHDPIB36(dendritic cell), pHDPCL05 (dendritic cell). Since none of these wasfound to encode a correctly spliced full-length form of this novelSiglec, a human spleen cDNA library in XZAPII (23) was screened with aKpnI-SacI fragment from HDPCL05. A single positive clone, p2.2, wasidentified and the phage insert subcloned into pBluescript andsequenced. Since clone 2.2 contained a correctly spliced full-lengthopen reading frame, the insert was subcloned into the mammalianexpression vector pcDNA3 (Invitrogen, Groningen, The Netherlands) andused as a template in subsequent molecular characterisation. Based onits sialic acid binding activity, this novel Siglec is hereon referredto as CD33-likeSV. To identify proteins related to CD33-likeSV, acomputer search of the GenBank nucleotide and proteinssequence databaseswas carried out using the Blast GeneSearch (National Center forBiotechnology Information, National Institutes of Health, Bethesda,Md.).

Phylogeny Analysis

Manipulations of sequences and alignments were performed using BaylorCollege of Medicine molecular biology software available on the internet(Human Genome Center, Baylor College of Medicine, Houston, Tex.).Protein and nucleotide alignments were performed using the SIM alignmentprogram (24). Chromosomal localisation Metaphase spreads prepared fromphytohemagglutinin-stimulated human lymphocytes were hybridized with abiotinylated 3.4 kb insert from pHEOMH 10 as described (25). Metaphasespreads from 50 cells were analysed. Northern blot analysis. Two humanMultiple Tissue Northern (MTN) blots containing approximately 2 μg ofpoly A⁺ RNA per lane from various human tissues were purchased fromClontech (Palo Alto, Calif.) and hybridised with ³²P-labelled insertfrom HEOMH10 as described previously (15).

Cells

The following cell lines were provided by the ICRF Cell ProductionService: COS-1, Balb/c 3T3 A3 1, Chinese hamster ovary KI (CHO), KG 1b,HL-60, U937, TBP-1 and Daudi. The NK-like cell line, YT, was obtainedfrom Dr Gillian Griffiths (Oxford University). Cells were cultured asdescribed previously (17). Human red blood cells (RBC) were obtainedfrom healthy donors and stored at 4° C. in Alsever's solution for up totwo weeks. Human blood leukocytes were obtained from whole blood bydextran sedimentation followed by lysis of contaminating RBC.Mononuclear fractions for flow cytometry were obtained by densitygradient centrifugation using Ficoll-Paque (Amersham Pharmacia Biotech).

Production of Fc-Proteins

Recombinant chimeras containing the entire extracellular region ofCD33-likeSV fused to the Fc region of human IgG1 (CD33-likeSV-Fc) wereprepared by PCR amplification of the entire extracellular region ofCD33-likeSV using the following forward and reverse primers (5′/3′):ACAAGCTTTGCGCCTCCTATGCGGAGATG and CCCCTCGAGCTTATCTGGCAGCTGCAGGAT. ThePCR product was cloned in-frame into the plGplus vector, which encodes aFactor Xa cleavage site between the extracellular region and the Fchinge region of human IgG1. CD33-likeSV-Fc. was produced in transientlytransfected COS cells and purified with protein A Sepharose as described(13).

Generation of Mouse Polyclonal and Monoclonal Antibodies to CD33-LikeSV

Balb/c 3T3 A31 cells were transfected by electroporation withCD33-likeSV cDNA in the pcDNA3 vector. G418 resistant clones expressingCD33-likeSV were identified by their ability to bind human RBC anddesignated CD33-likeSV-3T3. To generate polyclonal antibodies, Balb/cmice were immunized twice intraperitoneally, at an interval of 14 days,with 10⁷ live CD33-likeSV-3T3 cells. After a final boost, the immuneserum was collected, IgG purified by protein G Sepharose and passed overa CD33-likeSV-Fc column prepared by coupling 1.0 mg purifiedCD33-likeSV-Fc to cyanogen bromide activated Sepharose CL4B. Bound IgGwas eluted with 0.1 M glycine buffer pH 2.5 and neutralized with 0.1volumes of 0.1 M Tris pH 8.0. To generate a monoclonal antibody (mAb),Balb/c mice were immunised with CD33-likeSV-Fc and hybridomas generatedby fusing immune spleen cells with the SP2 myeloma following standardmethods (28). A positive well reacting specifically with CD33-likeSV-Fcwas identifed by enzyme-linked immunosorbent assays as described (21).The hybridoma was cloned three times by limiting dilution and the mAbdesignated 5G6 (IgGI). 5G6 was used as a tissue culture supernatant inall experiments.

Human RBC Binding Assays to CD33-LikeSV Expressed on CHO Cells and COSCells

CHO cells stably expressing CD33-likeSV (CD33-likeSV-CHO) were generatedby transfecting CHO cells with CD33-likeSV cDNA in the pcDNA3 vector.G418 resistant clones expressing CD33-likeSV were identified by theirability to bind the anti-CD33-likeSV mAb 5G6 and designatedCD33-likeSV-CHO. COS cells were transiently transfected with CD33-likeSVcDNA by electroporation. RBC binding assays with CD33-likeSV-transfectedCOS cells and CD33-likeSV-CHO cells were carried out as described (13).

Binding Assays with Polyacrylamide Glycoconjugates

COS-1 cells were transiently transfected by electroporation with cDNAsencoding CD33-likeSV or CR1 (CD35) as a negative control and bindingassays carried out 48-72 hours later, as described previously (17).Briefly, sialidase-treated and control cells were incubated withsaturating concentrations (20 gg/ml) of biotinylated 2,3 PAA or 2,6 PAAfor 1 h at room temperature, washed and incubated with ¹²⁵I-Streptavidinat 0.5 μCi/ml for 1 h at 4° C. Bound radioactivity was counted using aBeckman gamma-counter.

FACS Analysis

Single and double labelling of cells for flow cytometry were performedfollowing standard protocols (30). Following staining, cells were fixedin 2% formaldehyde and analysed on a Becton-Dickinson FACS analyser.

Immunoprecipitation

Wild-type CHO cells, CHO—CD33-likeSV and Daudi cells at 2×107/ml weresurface biotinylated and lysates prepared in 1% Triton-X-100.Immunoprecipitations were carried out following standard procedures (28)and precipitated material was run on 4-12% gradient SDS-PAGE gelsfollowed by western blotting on nitrocellulose. The blots were blockedand biotinylated proteins revealed using streptavidin-peroxidasefollowed by addition of the ECL reagent (Amersham Pharmacia Biotech).

Results and Discussion:

Characterization of CD33-LikeSV

21 unique expressed sequence tags encoding a novel Siglec-like proteinwere identified in 8 different human cDNA libraries, as follows(frequencies in parentheses): eosinophils (10), primary dendritic cells(6), bone marrow (2), unstimulated B cells (1) tonsils (1), spleen (1).A full-length clone encoding this novel sequence was isolated from ahuman spleen cDNA library. Based on sequence similarity with otherSiglecs (FIG. 37) and its ability to bind sialic acid (see below) thisprotein has been designated CD33-likeSV. The extracellular region ofCD33-likeSV contains a hydrophobic signal peptide and five Ig-likedomains that are made up of an N-terminal V-set domain and four C2 setdomains containing five potential N-linked glycosylation sites.Following the transmembrane region, there is a cytoplasmic tail of 126amino acids.

CD33-likeSV contains all of the characteristic features of the Siglecsubgroup of Ig superfamily proteins (FIG. 37). These include thecritical arginine at position 120 that has been shown in theligand-bound crystal structure of sialoadhesin to interact with thecarboxyl group of sialic acid (2). In addition, CD33-likeSV contains twoconserved aromatic residues, Phe²⁶ and Y¹³⁴ on the A and G strands ofthe V-set domain that for sialoadhesin are both tryptophans shown tomake hydrophobic interactions with the N-acetyl and glycerol side chainsof sialic acid (2). CD33-likeSV exhibits the unusual pattern ofcysteines in domains 1 and 2 that form the intra-sheet and inter-domaindisulfide bonds (2). Within the cytoplasmic tail there are threepotential tyrosine-based motifs. The most membrane proximal of these,LD⁵⁹⁷INV, fits the consensus sequence (Y(L/V/I)N(V/P)) for receptorsthat bind to the SH2 domain of the Grb2 (26-28). Grb2 is an adaptormolecule that is able to activate the Ras signaling pathway viainteractions with the Ras-specific guanine nucleotide exchange factor,Sos (29).

Although this motif has not been observed in CD33-related Siglecspreviously, a similar motif is present in CD22/Siglec-2 where it hasbeen shown capable of interacting with Grb2 following tyrosinephosphorylation of the CD22 cytoplasmic tail. The interaction of CD22with Grb2 was recently found to be important for the formation of aquaternary complex of CD22, SFHP, Grb2 and Shc implicated in thesignaling functions of CD22. The other two tyrosine-based motifs inCD33-likeSV are more typical of the CD33-related Siglec subset (FIG.37). The motif LHY⁶⁶⁷ ATL fits the ITIM consensus, (L/I/V/S)XYXX(L/V),and is similar to the corresponding motif in CD33/Siglec-3, LHYASL,which has been shown to be dominant in tyrosine phosphorylation andrecruitment of SHP-1 (4, 5, 9) and SHP-2 (4, 9). Similar to otherCD33-related Siglecs, the membrane distal motif, ADY⁶⁹¹ AEV does not fitthe ITIM consensus but is well-conserved amongst the Siglecs. Thecorresponding phosphopeptide for CD33 has been shown capable ofrecruiting SHP-2 but not SHP-1 (4, 9). The presence of several potentialtyrosine-based motifs in CD33-likeSV therefore indicates that thisprotein may be involved in signaling functions.

Similarity with Other CD33-Related Siglecs and Phylogenetic Analysis

Database searches showed that CD33-likeSV shares the highest sequencesimilarity with the CD33-related Siglec subset, namely Siglecs-3, -5,-6, -7, -8 and -9. Within the first two Ig-like domains, CD33-likeSV wasfound to be 40-48% identical to these proteins (FIG. 37). CD33-likeSV istherefore the least similar of the CD33-related Siglecs which have beenshown previously to share between 50 and 80% identity within the firsttwo Ig-like domains (15-19, 21). The database searches also identified ahuman chromosome 19 clone (CTD-2616J11) from the completed human genomesequence that contains the entire CD33-likeSV gene. This allowed us toaccurately map the intron-exon boundaries within the cDNA sequence (FIG.37). This revealed that, besides an additional Ig-like domain notrepresented in any of the other CD33-related Siglecs, CD33-likeSV has anextra linker region at the end of domain 3 encoded by a separate exon(FIG. 37). This linker is similar in length (16 amino acids) andsequence (63% identity at the nucleotide level) to the linker at the endof domain 2 which is present in all CD33-related Siglecs (FIG. 37).Since domains 3 and 4 of CD33-likeSV share 46% amino acid sequenceidentity, it seems likely that this region of the molecule arose bytandem duplication of two exons, encoding a linker and an associatedIg-like domain. Alignment of the CD33-likeSV amino acid sequence withother CD33-related Siglecs showed that domain 3 of Siglecs-5, -6, -7, -8and -9 is highly related to domain 4 of CD33-likeSV (50-67% identity).Domain 3 of Siglecs-5, -6, -7, -8 and -9 also aligned well with domain 3of CD33-likeSV (44-49% identity). This further supports the notion thatdomains 3 and 4 of CD33likeSV arose through a duplication process. Thealignment also showed that domain 5 of CD33-likeSV was highly related todomain 4 of Siglec-5 (71% identity).

To further investigate the possible evolutionary relationships betweenthe CD33-related Siglecs, unrooted phylogenetic trees were created usingboth the amino terminal regions (leader peptide, domain 1+domain 2) andcarboxy terminal regions (transmembrane region+cytoplasmic tails) of allCD33-related Siglecs (FIG. 38). Both phylogenetic trees gave verysimilar results (FIGS. 38A and 38B) and are therefore consistent withthe notion that CD33-likeSV represents the most ancient of theCD33-related subset, followed by Siglec-5. Based on the phylogeneticanalysis and the sequence comparisons, it is tempting to speculate thatCD33-likeSV gave rise to the other CD33-related Siglecs by a process ofsequential deletion, each involving a single deletion event (FIG. 39).According to this model, Siglec-5 could have arisen from CD33-likeSV bydeletion of the exons encoding domain 3 and its associated linker.Siglec-5 could have then have given rise to a three-domain Siglec (e.g.,Siglec-6) by a further deletion of Siglec-5 domain 4. The otherthree-domain, Siglecs (i.e., Siglecs-7,-8 & -9) could have then beengenerated by gene duplication events. This would be consistent with thehigh degree of sequence similarity shared between these three Siglecs(FIG. 1 & FIG. 2). In principle, CD33/Siglec-3 could have arisen fromany of the other CD33-related Siglecs by a single deletion event (FIG.3). It also seems likely based on sequence comparisons that NUG/Siglec 4and Siglec-5 are derived from a common progenitor, since they have thesame number of Ig domains and share low, but significant sequencesimilarity throughout the extracellular and intracellular regions (TableI).

Chromosomal Localization and Expression of the CD33-likeSV Gene

The CD33-likeSV gene was mapped by in situ hybridization to the long armof chromosome 19, in the 19q13.3 band (FIG. 40A), closely linked to theother CD33-related Siglecs (15-17, 19, 22, 30). Northern blot analysis(FIG. 40B) revealed presence of a major CD33-likeSV mRNA transcript ofapproximately 3.0 kb, with highest levels in spleen, lymph node, bloodleukocytes and appendix. Readily detectable mRNA could also be seen inseveral other tissues (FIG. 40B).

CD33-LikeSV Mediates Sialic Acid-Dependent Binding to Human RBC and toGlycoconjugates

To investigate the potential sialic acid binding properties ofCD33-likeSV, we initially performed binding assays in which native andsialidase-treated human RBC were added to transiently transfected COScells. Very little binding could be detected with untreated COS cellsexpressing CD33-likeSV. In contrast, following sialidase-treatment, highlevels of RBC binding were seen (data not shown). Similar results wereobtained using CHO cells stably transfected with CD33-likeSV (data notshown). Much of the increase in binding seen after treating Siglecexpressing cells with sialidase is thought to be due to unmasking thesialic acid binding site on Siglecs from cis interactions with sialicacids at the cell surface (14, 15, 17, 19, 21, 22, 31, 32). All bindingof RBC to sialidase-treated cells expressing CD33-likeSV was abolishedwhen the RBC were pretreated with sialidase, demonstrating that thebinding was sialic acid-dependent (data not shown).

To determine the sialic acid linkage preference of CD33-likeSV, bindingassays were carried out with synthetic polyacrylamide conjugates,carrying either 3′ or 6′ sialyllactose or lactose. In these experiments,COS cells were transiently transfected with CD33-likeSV or CR1 as anegative control. FACS analysis showed that 20-30% of the cellsexpressed each molecule three days after the transfection (data notshown). Transfected cells were either untreated or treated withsialidase immediately before the binding assay to remove potentiallyinhibitory sialic acids in the COS cell glycocalyx (FIG. 41). Verylittle binding of PAA conjugates could be observed with untreated cells,but after sialidase treatment, CD33-likeSV transfected COS cells boundstrongly to glycoconjugates carrying sialic acid in either α2,3 or α2,6linkages (FIG. 41). No specific binding was observed with lactose-PAAused as a negative control.

Expression of CD33-LikeSV on Human Peripheral Blood Leukocytes

An affinity purified mouse polyclonal antibody was prepared to theextracellular region of CD33-likeSV and shown by FACS analysis not tocross-react with Siglecs-3, -5, -7, and -8 expressed on CHO cells (datanot shown). A monoclonal antibody, 5G6, was also raised againstCD33-likeSV and shown not to cross-react with Siglecs-3, -5, -7, -8 and-9 expressed on CHO cells (data not shown). A detailed analysis of theexpression of CD33-likeSV was carried out by FACS analysis, using humanperipheral blood leukocytes. Indistinguishable results were obtainedusing either the polyclonal or monoclonal antibodies. First, expressionon granulocyte, monocyte, and lymphocyte subsets was compared (FIG.42A). This revealed low levels of CD33-likeSV on granulocytes andmonocytes and a subpopulation of lymphocytes. Since CD33-likeSVtranscripts were identified frequently in an eosinophil cDNA library, weasked whether CD33-likeSV was normally expressed on eosinophils. Usinganti CD16 mAb to distinguish eosinophils from neutrophils, theCD16-negative eosinophils were found to stain specifically with 5G6(FIG. 42B). To date, Siglec-8 is the only other Siglec that has beenshown to be expressed on eosinophils, although in this case, itsexpression is highly restricted (19,20).

To characterize the lymphocyte-reactive cells in more detail, doublelabeling was carried out in which staining for CD33-likeSV was combinedwith staining for CD3 (pan T cell), CD4 and CD8 (T cell subsets), CD19(pan B cell), CD16 (NK cells) and CD56 (NK cells) (FIG. 4B). Thedominant populations expressing CD33-likeSV were CD19⁺ B cells whichwere weakly labeled. Higher levels of CD33-likeSV was detected on a verysmall subset (0.5%) of cells in the lymphocyte gate that are alsostrongly positive and carry high levels of CD16 but no detectable CD56(FIG. 6C). A similar pattern of CD33-likeSV staining was observed in 8independent experiments with different donors, using either thepolyclonal or monoclonal antibody. In a previous study on the expressionof Siglec-9, the same population of CD16+CD56− cells was shown to belabeled at a similar level to the staining shown here with CD33-likeSV.Although these cells constitute only a very minor subset of the totalblood leukocytes, it is striking that they express two different Siglecsat relatively high levels. Currently the nature and potential functionsof these cells is unclear, but they may correspond to a minor populationof CD16+CD56− cells that were previously identified in human blood andshown to have the morphological features of NK cells and exhibit lowlevels of natural cytotoxicity. CD16+CD56− NK-like cells have been shownto be enriched in fetal cord blood compared with adult blood, and it hasbeen speculated that they may represent an earlier stage of NK celldifferentiation than the more normal CD16+CD56+ NK cells.

Finally, FACS staining of various human cell lines was also performed.Consistent with the staining pattern with blood leukocytes, weakpositive labeling was observed with the U937 promonocytic cell line andthe Daudi (B cell) line. No staining was seen with HL 60(myelomonocytic), THP-1 (monocytic) or YT (NK-like) cells (data notshown).

Molecular Characterisation of CD33-LikeSV

To investigate the molecular mass of CD33-likeSV expressed at the cellsurface, the protein was immunoprecipitated from CD33-likeSV-CHO cellsand from Daudi cells using the affinity purified polyclonal antibody(FIG. 43). In both cases, a single, heterogeneous band was observed ofapproximately 100 kDa under both reducing and non-reducing conditions,demonstrating that CD33-likeSV is exists as a monomer in the plasmamembrane (FIG. 43-gel No. 1). No material was precipitated fromwild-type CHO cells used as a negative control.

Conclusions:

The results presented here with CD33-likeSV extend our previous workcharacterizing novel Siglecs related to CD33/Siglec-3. Phylogenyanalysis and sequence comparisons indicate that CD33-likeSV is the mostancient of the CD33-related subset that may have given rise to the othermembers of the subset by exon deletion and gene duplication.

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It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Further, the hard copy of the sequence listing submitted herewith andthe corresponding computer readable form are both incorporated herein byreference in their entireties. Additionally, the specifications andsequence listings of U.S. Provisional Applications Nos. 60/243,792,filed on Oct. 30, 2000, 60/198,407, filed Apr. 19, 2000, and 60/105,971,filed Oct. 28, 1998, of U.S. application Ser. No. 09/836,353, filed Apr.18, 2001, and of International Application Serial No. PCT/US99/25031,filed Oct. 27, 1999, are all hereby incorporated by reference in theirentireties.

1. An isolated antibody or fragment thereof that specifically binds to aprotein selected from the group consisting of: (a) a protein whose aminoacid sequence consists of amino acid residues 23 to 1189 of SEQ IDNO:35; (b) a protein whose amino acid sequence consists of amino acidresidues 1 to 1189 of SEQ ID NO:35; (c) a protein whose amino acidsequence consists of a portion of SEQ ID NO:35, wherein said portion isat least 30 contiguous amino acid residues in length; and (d) a proteinwhose amino acid sequence consists of a portion of SEQ ID NO:35, whereinsaid portion is at least 50 contiguous amino acid residues in length. 2.The antibody or fragment thereof of claim 1 that specifically bindsprotein (a).
 3. The antibody or fragment thereof of claim 1 atspecifically binds protein (b).
 4. The antibody or fragment thereof ofclaim 1 that specifically binds protein (c).
 5. The antibody or fragmentthereof of claim 1 that specifically binds protein (d).
 6. The antibodyor fragment thereof of claim 3 wherein said protein bound by saidantibody or fragment thereof is glycosylated.
 7. The antibody orfragment thereof of claim 3 wherein said antibody or fragment thereof ishuman.
 8. The antibody or fragment thereof of claim 3 wherein saidantibody or fragment thereof is polyclonal.
 9. The antibody or fragmentthereof of claim 3 wherein said antibody or fragment thereof ismonoclonal.
 10. The antibody or fragment thereof of claim 3 which isselected from the group consisting of: (a) a chimeric antibody orfragment thereof; (b) a humanized antibody or fragment thereof; (c) asingle chain antibody; and (d) a Fab fragment.
 11. The antibody orfragment thereof of claim 3 which is labeled.
 12. The antibody orfragment thereof of claim 3 wherein said antibody or fragment thereofspecifically binds to said protein in a Western blot or an ELISA.
 13. Anisolated cell that produces the antibody or fragment thereof of claim 3.14. A hybridoma that produces the antibody or fragment thereof of claim3.
 15. A method of detecting an integrin alpha 11 protein, comprisingSEQ ID NO:35 in a biological sample comprising: (a) contacting thebiological sample with the antibody or fragment thereof claim 13; and(b) detecting the integrin alpha 11 protein in the biological sample.16. An isolated antibody or fragment thereof that specifically binds aprotein purified from a cell culture wherein said protein is encoded bya polynucleotide encoding amino acids 23 to 1189 of SEQ ID NO:35. 17.The antibody or fragment thereof of claim 16 wherein said antibody orfragment thereof is monoclonal.
 18. The antibody or fragment thereof ofclaim 16 wherein said antibody or fragment thereof is polyclonal. 19.The antibody or fragment thereof of claim 16 wherein said antibody orfragment thereof is human.
 20. The antibody or fragment thereof of claim16 which is selected from the group consisting of: (a) a chimericantibody or fragment thereof; (b) a humanized antibody or fragmentthereof; (c) a single chain antibody; and (d) a Fab fragment.
 21. Theantibody or fragment thereof of claim 16 wherein said antibody orfragment thereof specifically binds to said protein in a Western blot oran ELISA.
 22. The antibody or fragment thereof of claim 16 wherein theamino acid sequence of said protein consists of amino acid residues 1 to1189 of SEQ ID NO:35.